JP2024517956A - Chimeric antigen receptor T cell therapy - Google Patents

Abstract

The present disclosure provides methods of treating malignancies, including administering an effective dose of an immune cell therapy (e.g., chimeric antigen receptor gene modified T cell immunotherapy), and methods for manufacturing such immunotherapy. Some aspects of the present disclosure relate to methods of determining a patient's objective response to an immune cell immunotherapy based on the patient's levels and product attributes before and after administration of the immunotherapy to the patient.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims priority to U.S. Provisional Patent Application No. 63/188,916, filed May 14, 2021, No. 63/248,941, filed September 27, 2021, and No. 63/328,364, filed April 7, 2022, each of which is incorporated by reference in its entirety herein.

Human cancers are essentially composed of normal cells that have undergone genetic or epigenetic conversion to become abnormal cancer cells. In doing so, the cancer cells begin to express proteins and other antigens that differ from those expressed by normal cells. These abnormal tumor antigens can be used by the body's innate immune system to specifically target and kill cancer cells. However, cancer cells use various mechanisms to stop immune cells, such as T and B lymphocytes, from normally targeting them.

Human T cell therapy is based on human T cells that are enriched or modified to target and kill cancer cells in a patient. To increase the ability of T cells to target and kill specific cancer cells, methods have been developed to genetically engineer T cells to express constructs that direct the T cells to specific target cancer cells. Chimeric antigen receptors (CARs), which contain binding domains that can interact with specific tumor antigens, allow T cells to target and kill cancer cells that express the specific tumor antigen.

There is a need to understand how attributes of CAR-positive T cells and the patient's immunological status correlate with clinical outcomes of immunotherapy.

Provided herein are methods and uses of cells (e.g., engineered T cells) and/or compositions thereof for the treatment of subjects having a disease or condition that is or includes, generally, a cancer or tumor, e.g., leukemia or lymphoma. In some aspects, the methods and uses provide or achieve improved response and/or more durable response or efficacy and/or reduced risk of toxicity or other side effects in subjects treated with some methods compared to certain alternative methods. In some embodiments, the methods include administering a specific number or relative number of engineered cells, administering a predetermined ratio of specific types of cells, treating a specific patient population, such as a patient population with a specific risk profile, stage classification, and/or prior treatment history, administering additional therapeutic agents, and/or combinations thereof.

Also provided are methods that include evaluating a particular parameter following administration of a cell therapy, e.g., expression of a specific biomarker or analyte that may correlate with an outcome, e.g., a treatment outcome, including a response, such as a complete response (CR) or partial response (PR); or a safety outcome, such as toxicity, e.g., the development of neurotoxicity or CRS. Also provided are methods of assessing the likelihood of a response and/or the likelihood of a risk of toxicity based on evaluation of a parameter, e.g., expression of a biomarker or analyte.

In one aspect, the disclosure provides a method of treating a cancer expressing a tumor antigen in a subject in need of cancer treatment, comprising administering to the subject a therapeutically effective amount of CAR T cells expressing an antigen binding molecule that recognizes the tumor antigen. In some embodiments, the cancer is a leukemia or lymphoma. In some embodiments, the cancer is mantle cell lymphoma (MCL). In some embodiments, the MCL is relapsed/refractory after two or more systemic therapies. In some embodiments, the cancer is (relapsed/refractory) indolent non-Hodgkin's lymphoma (iNHL). In some embodiments, the cancer is follicular lymphoma (FL). In some embodiments, the cancer is marginal zone lymphoma (MZL). In some embodiments, the subject has MCL with high-risk features of progression within 24 months of initiation of a first anti-CD20-containing chemoimmunotherapy (MCL POD24). In some embodiments, the cancer is iNHL with high-risk features of disease progression within 24 months of diagnosis (iNHL POD24). In some embodiments, the tumor antigen is CD19. In some embodiments, the CAR T cell therapy is administered early. For example, the CAR-T cell therapy may be administered as a first line treatment and/or prior to progression.

The following embodiments are illustrative and non-limiting of the present disclosure.
1. A method for treating cancer, non-Hodgkin's lymphoma (NHL), comprising administering to a subject a therapeutically effective amount of immune cells directed against a tumor antigen.
2. The method of embodiment 1, wherein the subject is at high risk of disease progression.
3. The method of embodiment 1 or 2, wherein the NHL is mantle cell lymphoma (MCL) or indolent NHL (iNHL).
4. The method of embodiment 1 or 2, wherein the iNHL is marginal zone lymphoma (MZL) or follicular lymphoma (FL).
5. The method of embodiment 2, wherein the subject is at high risk if the subject exhibits disease progression within 24 months after initial diagnosis.
6. The method of embodiment 2, wherein the subject is at high risk if the subject exhibits disease progression within 24 months of a first anti-CD20-containing chemoimmunotherapy.
7. The method of embodiment 6, wherein the chemoimmunotherapy comprises an alkylating agent.
8. The method of any one of embodiments 1-7, wherein the immune cells are administered as a first, second, third, fourth, fifth, or sixth line treatment.
9. The method of any one of embodiments 1-8, wherein the immune cells are selected from tumor infiltrating lymphocytes (TIL), NK cells, autologous T cells, allogeneic T cells, and genetically engineered autologous T cells (eACT), and any combination thereof.
10. The method of embodiment 9, wherein the immune cell is a CAR T cell.
11. The method of embodiment 10, wherein the CAR T cell therapy comprises axicabtagene siloleucel or brexcabtagene autoleucel/KTE-X19.
12. The method of embodiment 1, 2, or 9, wherein the therapeutically effective amount, or effective dose, of immune cells is at least about ¹⁰⁴ cells, at least about ¹⁰⁵ cells, at least about ¹⁰⁶ cells, at least about ¹⁰⁷ cells, at least about ¹⁰⁸ cells, at least about ¹⁰⁹ cells, or at least about ¹⁰¹⁰ cells.
13. The method of embodiment 1, 2, or 9, wherein the therapeutically effective amount, or effective dose, of immune cells is about ¹⁰⁴ cells, about ¹⁰⁵ cells, about ¹⁰⁶ cells, about ¹⁰⁷ cells, or about ¹⁰⁸ cells.
14. The method of embodiment 1, 2, or 9, wherein the therapeutically effective amount, or effective dose, of the immune cells is about ^2x106 cells/kg, about ^3x106 cells/kg, about ^4x106 cells/kg, about ^5x106 cells/kg, about ^6x106 cells/kg, about ^7x106 cells/kg, about ^8x106 cells/kg, about ^9x106 cells/kg, about ^1x107 cells/kg, about ^2x107 cells/kg, about ^3x107 cells/kg, about ^4x107 cells/kg, about ^5x107 cells/kg, about ^6x107 cells/kg, about ^7x107 cells/kg, about ^8x107 cells/kg, or about ^9x107 cells/kg.
15. The method of embodiment 1, 2, or ⁹ , wherein the therapeutically effective amount, or effective dose, of immune cells is from about 1 x ¹⁰ to about ² x 10 immune cells per kg of body weight, up to about 1 x 10 immune cells per kg of body weight.
16. The method of embodiment 1, 2 or 9, wherein the therapeutically effective dose of immune cells is 75-200×10 ⁶ immune cells.
17. Tumor antigens include tumor-associated surface antigen, 5T4, alpha-fetoprotein (AFP), B7-1 (CD80), B7-2 (CD86), BCMA, B-human chorionic gonadotropin, CA-125, carcinoembryonic antigen (CEA), CD123, CD133, CD138, CD19, CD20, CD22, CD23, CD24, CD25, CD30, CD33, CD34, CD4, CD40, CD44, CD56, CD8, CLL-1, c-M et, CMV-specific antigen, CS-1, CSPG4, CTLA-4, DLL3, disialoganglioside GD2, ductal epithelial mucin, EBV-specific antigen, EGFR variant III (EGFRvIII), ELF2M, endoglin, ephrinB2, epidermal growth factor receptor (EGFR), epithelial cell adhesion molecule (EpCAM), epithelial tumor antigen, ErbB2 (HER2/neu), fibroblast-associated protein (fap), FLT3, folate-binding protein, GD2, GD3, glioma-associated antigen, glycosphingolipid, gp36, HBV-specific antigen, HCV-specific antigen, HER1-HER2, HER2-HER3 combination, HERV-K, high molecular weight melanoma-associated antigen (HMW-MAA), HIV-1 envelope glycoprotein gp41, HPV-specific antigen, human telomerase reverse transcriptase, IGF I receptor, IGF-II, IL-11Rα, IL-13R-a2, influenza virus-specific specific antigens; CD38, insulin growth factor-1 (IGF1), intestinal carboxylesterase, kappa chain, LAGA-1a, lambda chain, Lassa virus specific antigen, lectin-reactive AFP, lineage-specific antigens or tissue-specific antigens, e.g., CD3, MAGE, MAGE-A1, major histocompatibility complex (MHC) molecules, major histocompatibility complex (MHC) molecules presenting tumor-specific peptide epitopes, M-CSF, melanoma-associated antigen, mesothelin, MN-CA IX, MUC-1, mutant hsp70-2, mutant p53, mutant ras, neutrophil elastase, NKG2D, Nkp30, NY-ESO-1, p53, PAP, prostase, prostate specific antigen (PSA), prostate cancer tumor antigen-1 (PCTA-1), prostate specific antigen protein, STEAP1, STEAP2, PSMA, RAGE-1, ROR1, RU1, RU2 (AS), surface adhesion molecules, survivin and telomerase, TAG-72, fibronectin extra domain A (EDA) and extra domain B (EDB) and tenascin C A1 domain (TnC A1), thyroglobulin, tumor stromal antigen, vascular endothelial growth factor receptor-2 (VEGFR2), virus specific surface antigens, such as HIV specific antigens (e.g., HIV 17. The method of any one of embodiments 1-16, wherein the antigen is selected from the group consisting of the surface antigens IgG1, IgG2, IgG3, IgG4, IgG5, IgG6, IgG7, IgG8, IgG9, IgG10, IgG11, IgG12, IgG13, IgG14, IgG15, IgG16, IgG17, IgG18, IgG19, IgG109, IgG10
18. The method of embodiment 17, wherein the target antigen is CD19.
19. The method of embodiment 1 or 2, wherein the NHL is (relapsed or refractory) diffuse large B-cell lymphoma (DLBCL) - unspecified type, primary mediastinal large B-cell lymphoma, high-grade B-cell lymphoma, or DLBCL arising from follicular lymphoma.
20. The method of any one of embodiments 1-19, further comprising preconditioning the subject with one or more preconditioning agents.
21. The method of embodiment 20, wherein the subject is preconditioned with administration of an alkylating agent and/or a platinum-based agent.
22. The method of embodiment 21, wherein the alkylating agent is selected from the group consisting of melphalan, chlorambucil, cyclophosphamide, mechlorethamine, mustine (HN2), uramustine, uracil mustard, melphalan, chlorambucil, ifosfamide, bendamustine, carmustine, lomustine, streptozocin, alkyl sulfonates, busulfan, thiotepa or an analog thereof, and any combination thereof.
23. The method of embodiment 21, wherein the platinum-based preconditioning agent is selected from the group consisting of platinum, cisplatin, carboplatin, nedaplatin, oxaliplatin, satraplatin, triplatin tetranitrate, procarbazine, altretamine, triazene, dacarbazine, mitozolomide, temozolomide, dacarbazine, temozolomide, and any combination thereof.
24. The method of embodiment 20, wherein the preconditioning agents comprise cyclophosphamide and fludarabine.
25. The method of any one of embodiments 20-24, wherein administration of the one or more preconditioning agents begins at least 7 days, at least 6 days, at least 5 days, at least 4 days, at least 3 days, at least 2 days, or at least 1 day prior to administration of the cell therapy.

The following further embodiments are illustrative and non-limiting of the present disclosure.
1. A method of treating cancer in a subject in need thereof, wherein the cancer is non-Hodgkin's lymphoma (NHL) or relapsed/refractory B-cell precursor acute lymphoblastic leukemia or relapsed/refractory B-cell non-Hodgkin's lymphoma (R/R B-ALL), comprising administering to the subject a therapeutically effective amount of immune cells directed against a tumor antigen, optionally wherein the subject is a pediatric or adolescent subject.
2. The method of claim 1, wherein the subject is at high risk of disease progression.
3. The method of claim 1 or 2, wherein the NHL is mantle cell lymphoma (MCL) or indolent NHL (iNHL).
4. The method of claim 1 or 2, wherein the iNHL is marginal zone lymphoma (MZL) or follicular lymphoma (FL).
5. The method of claim 2, wherein the subject is at high risk if the subject exhibits disease progression within 24 months of initial diagnosis.
6. The method of claim 2, wherein the subject is at high risk if the subject exhibits disease progression within 24 months of a first anti-CD20-containing chemoimmunotherapy.
7. The method of claim 6, wherein the chemoimmunotherapy comprises an alkylating agent.
8. The method of any one of claims 1 to 7, wherein the immune cells are administered as a first, second, third, fourth, fifth, or sixth line treatment and/or prior to disease progression.
9. The method of any one of claims 1 to 8, wherein the immune cells are selected from tumor infiltrating lymphocytes (TIL), NK cells, autologous T cells, allogeneic T cells, and genetically engineered autologous T cells (eACT), and any combination thereof.
10. The method of claim 9, wherein the immune cells are CAR T cells.
11. The method of claim 10, wherein the CAR T cell therapy comprises axicabtagene siloleucel or brexcabtagene autoleucel/KTE-X-19.
12. The method of claim 1, 2, or 9, wherein the therapeutically effective amount, or effective dose, of the immune cells is at least about ¹⁰⁴ cells, at least about ¹⁰⁵ cells, at least about ¹⁰⁶ cells, at least about ¹⁰⁷ cells, at least about ¹⁰⁸ cells, at least about ¹⁰⁹ cells, or at least about ¹⁰¹⁰ cells.
13. The method of claim 1, 2, or 9, wherein the therapeutically effective amount, or effective dose, of the immune cells is about ¹⁰⁴ cells, about ¹⁰⁵ cells, about ¹⁰⁶ cells, about ¹⁰⁷ cells, or about ¹⁰⁸ cells.
14. The method of claim 1, 2, or 9, wherein the therapeutically effective amount, or effective dose, of the immune cells is about ^2x106 cells/kg, about ^3x106 cells/kg, about ^4x106 cells/kg, about ^5x106 cells/kg, about ^6x106 cells/kg, about ^7x106 cells/kg, about ^8x106 cells/kg, about ^9x106 cells/kg, about ^1x107 cells/kg, about ^2x107 cells/kg, about ^3x107 cells/kg, about ^4x107 cells/kg, about ^5x107 cells/kg, about ^6x107 cells/kg, about ^7x107 cells/kg, about ^8x107 cells/kg, or about ^9x107 cells/kg.
15. The method of claim 1, 2, or ⁹ , wherein the therapeutically effective amount, or effective dose, of the immune cells is from about 1 x ¹⁰ to about 2 x ¹⁰ immune cells per kg of body weight, up to about 1 x 10 immune cells per kg of body weight.
16. The method of claim 1, 2, or 9, wherein the therapeutically effective dose of immune cells is 75-200 x ¹⁰⁶ immune cells.
17. The tumor antigen is selected from the group consisting of tumor-associated surface antigen, 5T4, alpha-fetoprotein (AFP), B7-1 (CD80), B7-2 (CD86), BCMA, B-human chorionic gonadotropin, CA-125, carcinoembryonic antigen (CEA), CD123, CD133, CD138, CD19, CD20, CD22, CD23, CD24, CD25, CD30, CD33, CD34, CD4, CD40, CD44, CD56, CD8, CLL-1, c- Met, CMV-specific antigen, CS-1, CSPG4, CTLA-4, DLL3, disialoganglioside GD2, ductal epithelial mucin, EBV-specific antigen, EGFR variant III (EGFRvIII), ELF2M, endoglin, ephrinB2, epidermal growth factor receptor (EGFR), epithelial cell adhesion molecule (EpCAM), epithelial tumor antigen, ErbB2 (HER2/neu), fibroblast-associated protein (fap), FLT3, folate-binding protein , GD2, GD3, glioma-associated antigens, glycosphingolipids, gp36, HBV-specific antigens, HCV-specific antigens, HER1-HER2, HER2-HER3 combinations, HERV-K, high molecular weight melanoma-associated antigen (HMW-MAA), HIV-1 envelope glycoprotein gp41, HPV-specific antigens, human telomerase reverse transcriptase, IGF I receptor, IGF-II, IL-11Rα, IL-13R-a2, influenza virus-specific specific antigens; CD38, insulin growth factor-1 (IGF1), intestinal carboxylesterase, kappa chain, LAGA-1a, lambda chain, Lassa virus specific antigen, lectin-reactive AFP, lineage-specific antigens or tissue-specific antigens, e.g., CD3, MAGE, MAGE-A1, major histocompatibility complex (MHC) molecules, major histocompatibility complex (MHC) molecules presenting tumor-specific peptide epitopes, M-CSF, melanoma-associated antigen, mesothelin, MN-CA IX, MUC-1, mutant hsp70-2, mutant p53, mutant ras, neutrophil elastase, NKG2D, Nkp30, NY-ESO-1, p53, PAP, prostase, prostate specific antigen (PSA), prostate cancer tumor antigen-1 (PCTA-1), prostate specific antigen protein, STEAP1, STEAP2, PSMA, RAGE-1, ROR1, RU1, RU2 (AS), surface adhesion molecules, survivin and telomerase, TAG-72, extra domain A (EDA) and extra domain B (EDB) of fibronectin and A1 domain of tenascin-C (TnC A1), thyroglobulin, tumor stromal antigen, vascular endothelial growth factor receptor-2 (VEGFR2), virus specific surface antigens, such as HIV specific antigens (e.g., HIV gp120), as well as any derivative or variant of these surface antigens.
18. The method of claim 17, wherein the target antigen is CD19.
19. The method of claim 1 or 2, wherein the NHL is (relapsed or refractory) diffuse large B-cell lymphoma (DLBCL) - unspecified type, primary mediastinal large B-cell lymphoma, high-grade B-cell lymphoma, or DLBCL arising from follicular lymphoma.
20. The method of any one of claims 1 to 19, further comprising preconditioning the subject with one or more preconditioning agents.
21. The method of claim 20, wherein the subject is preconditioned with the administration of an alkylating agent and/or a platinum-based agent.
22. The method of claim 21, wherein the alkylating agent is selected from the group consisting of melphalan, chlorambucil, cyclophosphamide, mechlorethamine, mustine (HN2), uramustine, uracil mustard, melphalan, chlorambucil, ifosfamide, bendamustine, carmustine, lomustine, streptozocin, alkyl sulfonates, busulfan, thiotepa or an analog thereof, and any combination thereof.
23. The method of claim 21, wherein the platinum-based preconditioning agent is selected from the group consisting of platinum, cisplatin, carboplatin, nedaplatin, oxaliplatin, satraplatin, triplatin tetranitrate, procarbazine, altretamine, triazenes, dacarbazine, mitozolomide, temozolomide, dacarbazine, temozolomide, and any combination thereof.
24. The method of claim 20, wherein the preconditioning agents include cyclophosphamide and fludarabine.
25. The method of any one of claims 20-24, wherein the administration of the one or more preconditioning agents begins at least 7 days, at least 6 days, at least 5 days, at least 4 days, at least 3 days, at least 2 days, or at least 1 day prior to the administration of the cell therapy.
26. The method of claim 1, wherein the subject received allogeneic stem cell therapy (alloSCT) following treatment with anti-CD19 CAR T cell therapy.
27. The method of claim 1, wherein the subject has a high tumor burden.
28. The method of claim 1, wherein tocilizumab is administered for management of neurological events only in the setting of cytokine release syndrome and/or steroids are initiated for management of grade 2 neurological events.

The following further embodiments are illustrative and non-limiting of the present disclosure.

One embodiment of the present disclosure relates to a method of treating relapsed/refractory B-cell precursor acute lymphoblastic leukemia in a subject, the method comprising administering to the subject a therapeutically effective amount of immune cells against a tumor antigen, the subject being a pediatric or adolescent subject.

One embodiment of the present disclosure relates to the above method, wherein the therapeutically effective amount of immune cells is about 1×10 ⁶ to about 2×10 ⁶ immune cells per kg of body weight.

One embodiment of the present disclosure relates to the above method, in which the immune cells are administered in a total volume of about 40 mL to 68 mL.

One embodiment of the present disclosure relates to the above method, wherein the immune cells are administered in a total volume of about 40 mL.

One embodiment of the present disclosure relates to the above method, wherein the therapeutically effective amount of immune cells is about 1×10 ⁶ immune cells per kg of body weight.

One embodiment of the present disclosure relates to the above method, wherein the immune cells are administered as a first, second, third, fourth, fifth, or sixth line treatment, or prior to disease progression.

In one embodiment of the present disclosure, the antigen is a tumor-associated surface antigen, e.g., 5T4, alpha fetoprotein (AFP), B7-1 (CD80), B7-2 (CD86), BCMA, B-human chorionic gonadotropin, CA-125, carcinoembryonic antigen (CEA), CD123, CD133, CD138, CD19, CD20, CD22, CD23, CD24, CD25, CD30, CD33, CD34, CD4, CD40, CD44, CD56, CD8, CLL-1, c-Met, CMV-specific antigen, CS-1, CSPG4, CTLA-4, DLL3, disialoganglioside GD2, ductal epithelial mucin, EBV-specific antigen, EGFR variant III (EGFRvIII), ELF2M, endoglin, ephrinB2, epidermal growth factor receptor (EGFR), epithelial cell adhesion molecule (EpCAM), epithelial tumor antigen, ErbB2 (HER2/neu), fibroblast-associated protein (fap), FLT3, folate Binding proteins, GD2, GD3, glioma-associated antigens, glycosphingolipids, gp36, HBV-specific antigens, HCV-specific antigens, HER1-HER2, HER2-HER3 combinations, HERV-K, high molecular weight melanoma-associated antigen (HMW-MAA), HIV-1 envelope glycoprotein gp41, HPV-specific antigens, human telomerase reverse transcriptase, IGF I receptor, IGF-II, IL-11Rα, IL-13R-a2, influenza virus rus-specific antigens; CD38, insulin growth factor-1 (IGF1), intestinal carboxylesterase, kappa chain, LAGA-1a, lambda chain, Lassa virus-specific antigen, lectin-reactive AFP, lineage-specific antigens or tissue-specific antigens, e.g., CD3, MAGE, MAGE-A1, major histocompatibility complex (MHC) molecules, major histocompatibility complex (MHC) molecules presenting tumor-specific peptide epitopes, M-CSF, melanoma-associated antigen, mesothelin, MN-CA IX, MUC-1, mutant hsp70-2, mutant p53, mutant ras, neutrophil elastase, NKG2D, Nkp30, NY-ESO-1, p53, PAP, prostase, prostate specific antigen (PSA), prostate cancer tumor antigen-1 (PCTA-1), prostate specific antigen protein, STEAP1, STEAP2, PSMA, RAGE-1, ROR1, RU1, RU2 (AS), surface adhesion molecules, survivin and telomerase, TAG-72, extra domain A (EDA) and extra domain B (EDB) of fibronectin and A1 domain of tenascin-C (TnC A1), thyroglobulin, tumor stromal antigen, vascular endothelial growth factor receptor-2 (VEGFR2), virus specific surface antigens, such as HIV specific antigens (e.g., HIV gp120), as well as any derivative or variant of these surface antigens.

One embodiment of the present disclosure relates to the above method, in which the target antigen is CD19.

One embodiment of the present disclosure further comprises preconditioning the subject with one or more preconditioning agents, the one or more preconditioning agents being selected from at least one of an alkylating agent and a platinum-based agent, the alkylating agent being melphalan, chlorambucil, cyclophosphamide, mechlorethamine, mustine (HN2), uramustine, uracil mustard, melphalan, chlorambucil, ifosfamide, bendamustine, carmustine, lomustine, streptozotocin, The platinum-based preconditioning agent is selected from the group consisting of platinum, cisplatin, carboplatin, nedaplatin, oxaliplatin, satraplatin, triplatin tetranitrate, procarbazine, altretamine, triazene, dacarbazine, mitozolomide, temozolomide, dacarbazine, temozolomide, and any combination thereof.

One embodiment of the present disclosure relates to the above method, wherein the preconditioning agent comprises cyclophosphamide and fludarabine.

One embodiment of the present disclosure relates to the above method, wherein cyclophosphamide is administered at a dose of 200 mg/m ² /day to 2000 mg/m ² /day and fludarabine is administered at a dose of 20 mg/m ² /day to 900 mg/m ² /day.

One embodiment of the present disclosure relates to the above method, wherein administration of the one or more preconditioning agents begins at least 7 days, at least 6 days, at least 5 days, at least 4 days, at least 3 days, at least 2 days, or at least 1 day prior to administration of the immune cells.

One embodiment of the present disclosure relates to the above method, wherein the subject has a high tumor burden.

An embodiment of the present disclosure relates to the above method further comprising at least one of administering tocilizumab for management of neurological events only in the setting of cytokine release syndrome and administering a corticosteroid for management of grade 2 neurological events.

One embodiment of the present disclosure relates to the above method, wherein the subject is at high risk for disease progression, and the subject is at high risk if the subject exhibits disease progression within 24 months of initial diagnosis.

An embodiment of the present disclosure relates to the above method, wherein the immune cells are selected from tumor infiltrating lymphocytes (TIL), NK cells, autologous T cells, allogeneic T cells, and genetically engineered autologous T cells (eACT), and any combination thereof.

One embodiment of the present disclosure relates to the above method, in which the immune cells are CAR T cells.

One embodiment of the present disclosure relates to a method of treating cancer in a subject in need thereof, wherein the cancer is non-Hodgkin's lymphoma (NHL) or relapsed/refractory B-cell precursor acute lymphoblastic leukemia or relapsed/refractory B-cell non-Hodgkin's lymphoma (R/R B-ALL), comprising administering to the subject a therapeutically effective amount of immune cells directed against a tumor antigen, the immune cells being autologous T cells expressing an anti-CD19 chimeric antigen receptor (CAR).

One embodiment of the present disclosure relates to the above method, wherein the cancer is NHL, and the NHL is mantle cell lymphoma (MCL) or indolent NHL (iNHL).

One embodiment of the present disclosure relates to the above method, wherein the iNHL is marginal zone lymphoma (MZL) or follicular lymphoma (FL).

One embodiment of the present disclosure relates to the above method, wherein the cancer is NHL, and the NHL is (relapsed or refractory) diffuse large B-cell lymphoma (DLBCL) - unspecified type, primary mediastinal large B-cell lymphoma, high-grade B-cell lymphoma, or DLBCL arising from follicular lymphoma.

CLINICAL TRIAL-2 Clinical Trial Design: a. Administered after leukapheresis and completed ≥5 days prior to initiating conditioning chemotherapy. PET-CT was required postbridging. b. Bone marrow biopsy was to be performed at screening and if positive, not performed, or indeterminate, biopsy was required to confirm CR. AE, adverse event; CAR, chimeric antigen receptor; CR, complete response; CRS, cytokine release syndrome; IV, intravenous; MCL, mantle cell lymphoma; ORR, objective response rate; PO, oral; R/R, relapsed/refractory. IRRC-assessed ORR in patients with and without MCL POD24. Assessed by IRRC according to Lugano classification ^{7. a} One patient was not evaluable. CR, complete response; IRRC, independent radiological review committee; ORR, objective response rate; PD, progressive disease; for POD24, disease progression <24 months after initial diagnosis; for non-POD24, disease progression ≥24 months after initial diagnosis; PR, partial response; SD, stable disease. Duration of response (DR) (Figure 3A), progression-free survival (PFS) (Figure 3B), and overall survival (OS) (Figure 3C) by MCL POD24 status. ^a Among responding patients. DOR, duration of response; NE, not estimable; OS, overall survival; PFS, progression-free survival; POD24, disease progression <24 months after initial diagnosis; non-POD24, disease progression ≥24 months after initial diagnosis. Duration of response (DR) (Figure 3A), progression-free survival (PFS) (Figure 3B), and overall survival (OS) (Figure 3C) by MCL POD24 status. ^a Among responding patients. DOR, duration of response; NE, not estimable; OS, overall survival; PFS, progression-free survival; POD24, disease progression <24 months after initial diagnosis; non-POD24, disease progression ≥24 months after initial diagnosis. Duration of response (DR) (Figure 3A), progression-free survival (PFS) (Figure 3B), and overall survival (OS) (Figure 3C) by MCL POD24 status. ^a Among responding patients. DOR, duration of response; NE, not estimable; OS, overall survival; PFS, progression-free survival; POD24, disease progression <24 months after initial diagnosis; non-POD24, disease progression ≥24 months after initial diagnosis. CAR T cell expansion in MCL POD24 and non-MCL POD24 patients. Detectable B cells over time among patients with MCL POD24 and those without MCL POD24. CLINICAL TRIAL-5 Clinical Trial Design. ^aPatients with stable disease (without relapse) >1 year since completion of last treatment were not eligible. ^bSingle agent anti-CD20 antibodies did not count as treatment of choice for eligibility. ^cEfficacy evaluable patients included ≥80 FL-treated patients with ≥18 months follow-up after axi-cel infusion and MZL-treated patients with ≥4 weeks follow-up after axi-cel infusion as of the data cut-off date. axi-cel: axi-cabutagensilol-eucel; CAR: chimeric antigen receptor; FL: follicular lymphoma; iNHL: low-grade non-Hodgkin's lymphoma; IV: intravenous; mAb: monoclonal antibody; MZL: marginal zone lymphoma; POD24: disease progression is less than 24 months after starting initial anti-CD20-containing chemoimmunotherapy; R/R: relapsed/refractory. IRRC-assessed ORR in iNHL POD24 and non-iNHL POD24 patients. Assessed by IRRC according to the Lugano classification (Cheson BD, et al. J Clin Oncol. 2014;32:3059-68). ^aOf the 5 patients reported as ND, 4 (1 FL non-POD24, 3 MZL) were disease-free at baseline and post-baseline by the IRRC but were considered diseased by the investigator. One FL and POD24 patient died before the first disease assessment. CR, complete response; FL, follicular lymphoma; IRRC, independent radiological review committee; MZL, marginal zone lymphoma; ND, not performed/not defined; ORR, overall response rate; for iNHL POD24, disease progression is less than 24 months after starting initial anti-CD20-containing chemoimmunotherapy; PD, progressive disease; PR, partial response; SD, stable disease. DOR (Figure 8A), PFS (Figure 8B), and OS (Figure 8C) by iNHL POD24 status. DOR, duration of response; FL, follicular lymphoma; mo, months; MZL, marginal zone lymphoma; NE, not estimable; NR, not reached; OS, overall survival; PFS, progression-free survival; for POD24, disease progression is less than 24 months after starting initial anti-CD20-containing chemoimmunotherapy. DOR (Figure 8A), PFS (Figure 8B), and OS (Figure 8C) by iNHL POD24 status. DOR, duration of response; FL, follicular lymphoma; mo, months; MZL, marginal zone lymphoma; NE, not estimable; NR, not reached; OS, overall survival; PFS, progression-free survival; for POD24, disease progression is less than 24 months after starting initial anti-CD20-containing chemoimmunotherapy. DOR (Figure 8A), PFS (Figure 8B), and OS (Figure 8C) by iNHL POD24 status. DOR, duration of response; FL, follicular lymphoma; mo, months; MZL, marginal zone lymphoma; NE, not estimable; NR, not reached; OS, overall survival; PFS, progression-free survival; for POD24, disease progression is less than 24 months after starting initial anti-CD20-containing chemoimmunotherapy. CAR T cell expansion (FIG. 9A and FIG. 9B) and pre-treatment serum analytes (FIG. 9C) in FL patients by iNHL POD24 status. P values were calculated using Wilcoxon rank sum test. ^aData were not available for two FL patients prior to retreatment. AUC _0-28 , area under the curve from day 0 to day 28, CAR, chimeric antigen receptor, CCL, chemokine (C-C motif) ligand, FL, follicular lymphoma, LOQ, limit of quantification, MDC, macrophage-derived chemokine, for POD24, disease progression was less than 24 months after starting initial anti-CD20-containing chemoimmunotherapy, and TARC, thymus and activation-regulated chemokine. CAR T cell proliferation (FIG. 9A and FIG. 9B) and pre-treatment serum analytes (FIG. 9C) in FL patients by iNHL POD24 status. P values were calculated using Wilcoxon rank sum test. ^aData were not available for two FL patients prior to retreatment. AUC _0-28 , area under the curve from day 0 to day 28, CAR, chimeric antigen receptor, CCL, chemokine (C-C motif) ligand, FL, follicular lymphoma, LOQ, limit of quantification, MDC, macrophage-derived chemokine, for POD24, disease progression was less than 24 months after starting initial anti-CD20-containing chemoimmunotherapy, and TARC, thymus and activation-regulated chemokine. CAR T cell proliferation (FIG. 9A and FIG. 9B) and pre-treatment serum analytes (FIG. 9C) in FL patients by iNHL POD24 status. P values were calculated using Wilcoxon rank sum test. ^aData were not available for two FL patients prior to retreatment. AUC _0-28 , area under the curve from day 0 to day 28, CAR, chimeric antigen receptor, CCL, chemokine (C-C motif) ligand, FL, follicular lymphoma, LOQ, limit of quantification, MDC, macrophage-derived chemokine, for POD24, disease progression was less than 24 months after starting initial anti-CD20-containing chemoimmunotherapy, and TARC, thymus and activation-regulated chemokine. Associations between AUC of CAR gene copy number/μg DNA (FIG. 10A), response (FIG. 10B), MRD rate (FIG. 10C), and toxicity (FIG. 10D and FIG. 10E). Associations between AUC of CAR gene copy number/μg DNA (FIG. 10A), response (FIG. 10B), MRD rate (FIG. 10C), and toxicity (FIG. 10D and FIG. 10E). Associations between AUC of CAR gene copy number/μg DNA (FIG. 10A), response (FIG. 10B), MRD rate (FIG. 10C), and toxicity (FIG. 10D and FIG. 10E). Associations between AUC of CAR gene copy number/μg DNA (FIG. 10A), response (FIG. 10B), MRD rate (FIG. 10C), and toxicity (FIG. 10D and FIG. 10E). Associations between AUC of CAR gene copy number/μg DNA (FIG. 10A), response (FIG. 10B), MRD rate (FIG. 10C), and toxicity (FIG. 10D and FIG. 10E). Additional cytokine and inflammatory marker levels over time: CRP, C-reactive protein; CXCL10, C-X-C motif chemokine ligand 10; GM-CSF, granulocyte-macrophage colony-stimulating factor; IL, interleukin; MCP, monocyte-attractant protein; Rα, receptor alpha; RA, receptor antagonist; SAA, serum amyloid A; VCAM, vascular cell adhesion molecule. Subgroup analysis of overall remission rate. BM, bone marrow; ORR, overall remission rate; SCT, stem cell transplant. Subgroup analysis of overall remission rate. BM, bone marrow; ORR, overall remission rate; SCT, stem cell transplant.

DEFINITIONS In order that this disclosure may be more readily understood, certain terms are first defined below. Additional definitions for the following terms, as well as other terms, are found throughout the specification.

As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

As used herein, unless otherwise stated or clear from the context, the term "or" is understood to be inclusive and includes both "or" and "and."

The term "and/or" as used herein should be construed as a specific disclosure of each of the two specified features or components, with or without the other. Thus, the term "and/or" as used herein in phrases such as "A and/or B" is intended to include A and B, A or B; A (alone); and B (alone). Similarly, the term "and/or" as used in phrases such as "A, B, and/or C" is intended to include each of the following aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

As used herein, the terms "for example" and "i.e." are used merely as examples, are not intended to be limiting, and should not be construed as referring only to the items explicitly listed herein.

Terms such as "more than," "at least," and "more than," e.g., "at least one," are intended to mean, but are not limited to, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 4, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 10 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, or 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, or more than the recited values. Any larger number or fraction in between is also included.

Conversely, the term "less than" includes each value less than the recited value. For example, "100 nucleotides or less" includes 100, 99, 98, 97, 96, 95, 94, 93, 92, 91, 90, 89, 88, 87, 86, 85, 84, 83, 82, 81, 80, 79, 78, 77, 76, 75, 74, 73, 72, 71, 70, 69, 68, 67, 66, 65, 64, 63, 62, 61, 60, 59, 58, 57, 56, 55, 54, 5 Included are 3, 52, 51, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, and 0 nucleotides. Any smaller number or fraction in between is also included.

Terms such as "multiple," "at least two," "two or more," and "at least a second" are not intended to be limiting, but include at least, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63 , 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 10 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, or 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, or more. Any larger number or fraction in between is also included.

Throughout this specification, the word "comprising" or variations such as "comprises" or "comprising" are understood to mean the inclusion of the recited elements, integers or steps, or group of elements, integers or steps, but not the exclusion of any other elements, integers or steps, or group of elements, integers or steps. Whenever an embodiment is described herein with the term "comprising," it is understood that other similar embodiments described with the terms "consisting of" and/or "consisting essentially of" are also presented. The term "consisting of" excludes any element, step, or ingredient not specified in the claim. In re Gray, 53 F. 2d 520, 11 USPQ 255 (CCPA 1931); Experte Davis, 80 USPQ 448,450 (Bd. App. 1948) ("consisting of" is defined as "closing the claim to the inclusion of materials other than those recited except for impurities ordinarily associated therewith"). The term "consisting essentially of" limits the claim to the materials or steps specified and those which "do not materially affect the basic and novel characteristics" of the claimed patent.

As used herein, unless otherwise specified or clear from the context, the term "about" refers to a value or composition that is within an acceptable error range of a particular value or composition as determined by one of ordinary skill in the art, which depends in part on how the value or composition is measured or determined, i.e., the limitations of the measurement system. For example, "about" or "approximately" can mean within one or more standard deviations, as per convention in the art. "About" or "approximately" can mean a range of up to 10% (i.e., ±10%). Thus, "about" can be understood to be 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, 0.01%, or 0.001% greater or less than the specified value. For example, about 5 mg can encompass any amount between 4.5 mg and 5.5 mg. Additionally, particularly with respect to biological systems or processes, the term can mean up to an order of magnitude or up to 5 times the value. When a particular value or composition is presented in this disclosure, unless otherwise specified, the meaning of "about" or "approximately" should be assumed to be within the acceptable error range of that particular value or composition.

As described herein, any concentration range, percentage range, ratio range, or integer range should be understood to include any integer value within the recited range, and fractions thereof, as appropriate (such as tenths and hundredths of integers), unless otherwise specified.

Units, prefixes and symbols used herein are presented using the format accepted by the Système International des Unites (SI). Numeric ranges are inclusive of the numbers defining the range.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. See, e.g., Juo, "The Concise Dictionary of Biomedicine and Molecular Biology", 2nd ed., (2001), CRC Press; "The Dictionary of Cell & Molecular Biology", 5th ed., (2013), Academic Press; and "The Oxford Dictionary of Biochemistry and Molecular Biology", Cammack et al. eds., 2nd ed, (2006), Oxford University Press, provides those of skill in the art with a general dictionary of many of the terms used in this disclosure.

"Administering" refers to the physical introduction of an agent into a subject using any of a variety of methods and delivery systems known to those of skill in the art. Exemplary routes of administration for the formulations disclosed herein include intravenous, intramuscular, subcutaneous, intraperitoneal, spinal or other parenteral routes of administration, for example, by injection or infusion. Exemplary routes of administration for the compositions disclosed herein include intravenous, intramuscular, subcutaneous, intraperitoneal, spinal or other parenteral routes of administration, for example, by injection or infusion. As used herein, the phrase "parenteral administration" refers to modes of administration other than enteral and topical administration, usually by injection, and includes, but is not limited to, intravenous, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injection and infusion, and in vivo electroporation. In some embodiments, the formulation is administered via a parenteral route, e.g., orally. Other non-parenteral routes include topical, epidermal, or mucosal routes of administration, e.g., intranasal, intravaginal, rectal, sublingual, or topical. Administration may also be performed, e.g., once, multiple times, and/or over one or more extended periods of time. In one embodiment, the CAR T cell therapy is administered via an "infusion product" that includes the CAR T cells.

The term "antibody" (Ab) includes, but is not limited to, a glycoprotein immunoglobulin that specifically binds to an antigen. In general, an antibody may include at least two heavy (H) chains and two light (L) chains, or antigen-binding molecules thereof, interlinked by disulfide bonds. Each H chain includes a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region includes three constant domains, CH1, CH2, and CH3. Each light chain includes a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region includes one constant domain, CL. The VH and VL regions may be further subdivided into hypervariable regions called "complementarity determining regions" (CDRs), separated by more conserved regions called "framework regions" (FRs). Each VH and VL contains three CDRs and four FRs arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant regions of Abs may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system.

Examples of antibodies include monoclonal antibodies, recombinantly produced antibodies, monospecific antibodies, multispecific antibodies (including bispecific antibodies), human antibodies, engineered antibodies, humanized antibodies, chimeric antibodies, immunoglobulins, synthetic antibodies, tetrameric antibodies comprising two heavy chain and two light chain molecules, antibody light chain monomers, antibody heavy chain monomers, antibody light chain dimers, antibody heavy chain dimers, antibody light chain-antibody heavy chain pairs, intracellular antibodies, antibody fusions (sometimes referred to herein as "antibody conjugates"), heteroconjugate antibodies, single domain antibodies, monovalent antibodies, single chain antibodies or single-chain Fvs (single-chain Fvs). Fv, scFv), camelized antibodies, affibodies, Fab fragments, F(ab')2 fragments, disulfide-linked Fv (sdFv), anti-idiotypic (anti-Id) antibodies (including, for example, anti-anti-Id antibodies), minibodies, domain antibodies, synthetic antibodies (sometimes referred to herein as "antibody mimetics"), and antigen-binding fragments of any of the above. In some embodiments, the antibodies described herein refer to polyclonal antibody populations.

An "antigen-binding molecule," "antigen-binding portion," or "antibody fragment" refers to any molecule that contains an antigen-binding portion (e.g., CDR) of the antibody from which the molecule is derived. An antigen-binding molecule may contain an antigen complementarity determining region (CDR). Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments, dAbs, linear antibodies, scFv antibodies, and multispecific antibodies formed from antigen-binding molecules. Peptibodies (i.e., Fc fusion molecules containing a peptide binding domain) are another example of a suitable antigen-binding molecule. In some embodiments, the antigen-binding molecule binds to an antigen on a tumor cell. In some embodiments, the antigen-binding molecule binds to an antigen on a cell involved in a hyperproliferative disease or to a viral or bacterial antigen. In some embodiments, the antigen-binding molecule binds to CD19. In further embodiments, the antigen-binding molecule is an antibody fragment that specifically binds to an antigen, including one or more of its complementarity determining regions (CDRs). In further embodiments, the antigen-binding molecule is a single chain variable fragment (scFv). In some embodiments, the antigen-binding molecule comprises or consists of an avimer.

"Antigen" refers to any molecule capable of eliciting an immune response or being bound by an antibody or antigen-binding molecule. The immune response may involve either antibody production or activation of specific immunocompetent cells, or both. One of skill in the art will readily appreciate that any macromolecule may function as an antigen, including virtually any protein or peptide. Antigens may be expressed endogenously, i.e., by genomic DNA, or may be expressed recombinantly. Antigens may be specific to a particular tissue, such as cancer cells, or may be broadly expressed. Additionally, fragments of larger molecules may act as antigens. In some embodiments, the antigen is a tumor antigen.

The term "neutralizing" refers to an antigen-binding molecule, scFv, antibody, or fragment thereof that binds to a ligand and prevents or reduces the biological action of that ligand. In some embodiments, the antigen-binding molecule, scFv, antibody, or fragment thereof directly blocks a binding site on the ligand or otherwise alters the ability of the ligand to bind by indirect means (such as a structural or energetic change in the ligand). In some embodiments, the antigen-binding molecule, scFv, antibody, or fragment thereof prevents the protein to which it is bound from performing a biological function.

The term "autologous" refers to any material derived from the same individual that is later reintroduced. For example, the method of genetically engineered autologous cell therapy (eACT™) described herein involves harvesting lymphocytes from a patient, which are then engineered to express, for example, a CAR construct, and then administered to the same patient.

The term "allogeneic" refers to any material derived from one individual and then introduced into another individual of the same species, e.g., allogeneic T cell transplantation.

In one embodiment, the CAR T cell therapy comprises an "axicabtagene silol eucel therapy." The "axicabtagene silol eucel therapy" consists of a single infusion of autologous anti-CD19 CAR-transduced T cells administered intravenously at a target dose of 2×10 ⁶ anti-CD19 CAR T cells/kg. In subjects weighing more than 100 kg, a maximum fixed dose of 2×10 ⁸ anti-CD19 CAR T cells may be administered. Anti-CD19 CAR T cells are autologous human T cells genetically engineered to express an extracellular single chain variable fragment (scFv) with specificity for CD19 linked to a tandem intracellular signaling moiety consisting of signaling domains derived from CD28 and CD3ζ (CD3 zeta) molecules. The anti-CD19 CAR vector construct was designed, optimized, and initially tested at the Department of Surgery of the National Cancer Institute (National Cancer Institute, NCI, IND 13871) (Kochenderfer et al, J Immunother. 2009;32(7):689-702; Kochenderfer et al, Blood. 2010;116(19):3875-86). The scFv is derived from the variable region of the anti-CD19 monoclonal antibody FMC63 (Nicholson et al, Molecular Immunology. 1997;34(16-17):1157-65). The CD28 co-stimulatory molecule fragment is added because mouse models suggest that it is important for the antitumor efficacy and persistence of anti-CD19 CAR T cells (Kowolik et al, Cancer Res. 2006;66(22):10995-1004). The signaling domain of the CD3 ζ chain is used for T cell activation. These fragments were cloned into a murine stem cell virus-based vector (MSGV1) and utilized to engineer autologous T cells. The CAR construct is inserted into the genome of T cells by retroviral vector transduction. Briefly, peripheral blood mononuclear cells (PBMCs) are obtained by leukapheresis and Ficoll separation. The PBMCs are activated by culturing with anti-CD3 antibodies in the presence of recombinant interleukin 2 (IL-2). The stimulated cells are transduced with a retroviral vector containing the anti-CD19 CAR gene and expanded in culture to generate sufficient engineered T cells for administration. Axicabtagene Siloleucel is a subject-specific product.

In one embodiment, the CAR T cell therapy comprises KTE-X19, an autologous anti-CD19 chimeric antigen receptor (CAR) T cell therapy approved in the United States and European Union for the treatment of relapsed/refractory (R/R) MCL (TECARTUS® (brexcavtagene autreucel) Prescribing Information. Kite Pharma, Inc; 2021; TECARTUS® (autologous anti-CD19 transduced CD3+ cells) Summary of Product Characteristics. Kite Pharma EU B.V.; 2021). The manufacturing process for KTE-X19 was modified for axicabtagene siloleucel to eliminate circulating lymphoma cells by positive enrichment of CD4 ⁺ /CD8 ⁺ cells.

In one embodiment, the product is characterized with respect to cellular composition. Cells may be labeled with fluorescently conjugated antibodies against CD3 (pan T cell marker), CD14, CD19 (B cell marker), CD45 (pan leukocyte marker), and CD56 (activation and NK marker) and assessed by flow cytometry. Cell viability may be assessed using Viability Dye (SYTOX, near IR) dead cell stain. The lower limit of quantification (LLOQ) of the assay may be 0.2% and 5% for NK cells and monocytes. The percentage of NK cells may be determined (NK cells were CD45 ⁺ , CD14 ⁻ , CD3 ⁻ , and CD56 ⁺ , T cells were CD45 ⁺ , CD14 ⁻ , and CD3 ⁻ ). The median percentage of NK cells for 23 lots of Axicabtagene Siloleucel and 97 lots of KTE-X19 can be 1.9% (range, 0.8%-3.2%) and 0.1% (range, 0.0%-2.8%), respectively. The median percentage of CD3 ⁻ cell impurities for the same lots of Axicabtagene Siloleucel and KTE-X19 can be 2.4% (range, 0.9%-4.6%) and 0.5% (range, 0.3%-3.9%), respectively. The cell viability of KTE-X19 (Brexcavtagene Autorucel, TECARTUS) and Axicabtagene Siloleucel (YESCARTA) may be 72% or more and 80% or more, respectively, anti-CD19 CAR expression may be 24% or more and 15% or more, respectively, IFN-γ production may be 190pg/mL or more and 520pg/mL or more, respectively, and the percentage of CD3 ⁺ cells may be 90% or more and 85% or more, respectively. Brexcavtagene Autorucel may be composed primarily of CD3+ T cells (99.3%±0.8%), which may be further classified into CD4+ (37.9%±16.5%) and CD8+ (59.3%±16.5%) subsets.

The terms "transduction" and "transduced" refer to the process by which foreign DNA is introduced into a cell by a viral vector (see Jones et al., "Genetics: principles and analysis," Boston: Jones & Bartlett Publ. (1998)). In some embodiments, the vector is a retroviral vector, a DNA vector, an RNA vector, an adenoviral vector, a baculoviral vector, an Epstein-Barr virus vector, a papovavirus vector, a vaccinia virus vector, a herpes simplex virus vector, an adenovirus-associated vector, a lentiviral vector, or any combination thereof.

"Cancer" refers to a broad group of various diseases characterized by the uncontrolled growth of abnormal cells in the body. Uncontrolled cell division and proliferation leads to the formation of malignant tumors that may invade adjacent tissues and metastasize to distant parts of the body via the lymphatic system or bloodstream. "Cancer" or "cancerous tissue" may encompass tumors. In this application, the term cancer is synonymous with malignant tumor. Examples of cancers that may be treated by the methods disclosed herein include cancers of the immune system, including, but not limited to, lymphomas, leukemias, myelomas, and other white blood cell malignancies. In some embodiments, the methods disclosed herein are directed to treating a variety of cancers, including, for example, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, cutaneous or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, testicular cancer, uterine cancer, cancer of the fallopian tubes, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, multiple myeloma, Hodgkin's disease, non-Hodgkin's lymphoma (NHL), primary mediastinal large B-cell lymphoma (PMBC), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), transformed follicular lymphoma, splenic marginal zone lymphoma (SMZL), esophageal cancer, small intestine cancer, cancer of the endocrine system, thyroid cancer, cancer of the parathyroid gland, adrenal gland cancer, soft tissue cancer, and the like. It may be used to reduce tumor size in tumors resulting from sarcoma, urethral cancer, penile cancer, chronic or acute leukemia, acute myeloid leukemia, chronic myelogenous leukemia, acute lymphoblastic leukemia (ALL) (including non-T-cell ALL), chronic lymphocytic leukemia (CLL), childhood solid tumors, lymphocytic lymphoma, bladder cancer, kidney or ureter cancer, renal pelvis cancer, central nervous system (CNS) neoplasms, primary CNS lymphoma, tumor angiogenesis, spinal axis tumor, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, environmentally induced cancers including those induced by asbestos, other B-cell malignancies, and combinations of the above cancers. In some embodiments, the cancer is multiple myeloma. In some embodiments, the cancer is NHL. Certain cancers may be responsive to chemotherapy or radiation therapy, or certain cancers may be resistant to therapy. A refractory cancer refers to a cancer that is not amenable to surgical intervention; either the cancer is initially non-responsive to chemotherapy or radiation therapy, or the cancer becomes non-responsive over time.

As used herein, "anti-tumor effect" refers to a biological effect that may be manifested as a reduction in tumor volume, a reduction in tumor cell number, a reduction in tumor cell proliferation, a reduction in the number of metastases, an increase in overall survival or progression-free survival, an increase in life expectancy, or an improvement in various physiological symptoms associated with a tumor. An anti-tumor effect may also refer to the prevention of tumor development, e.g., a vaccine.

As used herein, "cytokine" refers to a non-antibody protein released by a cell in response to contact with a specific antigen, where the cytokine interacts with another cell to mediate a response in the other cell. As used herein, "cytokine" is intended to refer to a protein released by a cell population that acts on another cell as an intercellular mediator. Cytokines can be expressed endogenously by a cell or can be administered to a subject. Cytokines can be released by immune cells, such as macrophages, B cells, T cells, and mast cells, to propagate an immune response. Cytokines can induce a variety of responses in recipient cells. Cytokines can include homeostatic cytokines, chemokines, proinflammatory cytokines, effectors, and acute phase proteins. Homeostatic cytokines, including, for example, interleukin (IL) 7 and IL-15, promote survival and proliferation of immune cells, and proinflammatory cytokines can promote an inflammatory response. Examples of homeostatic cytokines include, but are not limited to, IL-2, IL-4, IL-5, IL-7, IL-10, IL-12p40, IL-12p70, IL-15, and interferon (IFN) gamma. Examples of proinflammatory cytokines include, but are not limited to, IL-1a, IL-1b, IL-6, IL-13, IL-17a, tumor necrosis factor (TNF)-alpha, TNF-beta, fibroblast growth factor (FGF) 2, granulocyte macrophage colony-stimulating factor (GM-CSF), soluble intercellular adhesion molecule 1 (sICAM-1), soluble vascular adhesion molecule 1 (sVCAM-1), vascular endothelial growth factor (VEGF), VEGF-C, VEGF-D, and placental growth factor (PLGF). Examples of effectors include, but are not limited to, granzyme A, granzyme B, soluble Fas ligand (sFasL), and perforin. Examples of acute phase proteins include, but are not limited to, C-reactive protein (CRP) and serum amyloid A (SAA).

A "chemokine" is a type of cytokine that mediates chemotaxis or directional movement of cells. Examples of chemokines include, but are not limited to, IL-8, IL-16, eotaxin, eotaxin-3, macrophage-derived chemokine (MDC or CCL22), monocyte chemoattractant protein 1 (MCP-1 or CCL2), MCP-4, macrophage inflammatory protein 1 alpha (MIP-1α, MIP-1a), MIP-1β (MIP-1b), gamma-inducible protein 10 (IP-10), and thymus and activation-regulated chemokine (TARC or CCL17).

As used herein, "chimeric receptor" refers to a surface-expressed engineered molecule capable of recognizing a specific molecule. Chimeric antigen receptors (CARs) and engineered T cell receptors (TCRs) that contain a binding domain capable of interacting with a specific tumor antigen allow T cells to target and kill cancer cells expressing a specific tumor antigen. In one embodiment, T cell therapy is based on T cells engineered to express a chimeric antigen receptor (CAR) or T cell receptor (TCR) that contains (i) an antigen binding molecule, (ii) a costimulatory domain, and (iii) an activation domain. The costimulatory domain may include an extracellular domain, a transmembrane domain, and an intracellular domain, where the extracellular domain includes a hinge domain that may be truncated.

A "therapeutically effective amount," "effective dose," "effective amount," or "therapeutically effective dosage" of a therapeutic agent, e.g., engineered CAR T cells, small molecules, "agents" described herein, is any amount that, when used alone or in combination with another therapeutic agent, protects a subject from developing a disease or promotes regression of the disease as evidenced by a decrease in the severity of disease symptoms, an increase in the frequency and duration of symptom-free periods of the disease, or prevention of functional impairment or disability due to the affliction of the disease. Such terms may be used interchangeably. The ability of a therapeutic agent to promote regression of a disease may be evaluated using a variety of methods known to those of skill in the art, for example, by analyzing the activity of the agent in human subjects during clinical trials, in animal model systems predictive of efficacy in humans, or in in vitro assays. Therapeutically effective amounts and dosing regimens may be empirically determined by testing in known in vitro or in vivo (e.g., animal model) systems.

The term "combination" refers to either a fixed combination in one unit dosage form, or a combined administration in which the compound of the present disclosure and a combination partner (e.g., another drug, also referred to as a "therapeutic agent" or "drug", as described below) can be administered independently at the same time or separately within time intervals, particularly where these time intervals allow the combination partners to exhibit a cooperative effect, e.g., a synergistic effect. The individual components can be packaged in a kit or packaged separately. One or both of the components (e.g., powder or liquid) can be reconstituted or diluted to the desired dose before administration. As used herein, the terms "co-administration" or "co-administration" and the like are intended to include administration to a single subject (e.g., patient) in need of administration of the selected combination partner, and are intended to encompass treatment regimens in which the agents are not necessarily administered by the same route of administration or at the same time.

The terms "product" or "infusion product" are used interchangeably herein and refer to a T cell composition administered to a subject in need thereof. Typically, in CAR T cell therapy, the T cell composition is administered as an infusion product.

As used herein, the term "lymphocyte" includes natural killer (NK) cells, T cells, or B cells. NK cells are a type of cytotoxic (cell-toxic) lymphocyte that is a major component of the innate immune system. NK cells reject tumor and virus-infected cells. They act through the process of apoptosis, or programmed cell death. They were called "natural killers" because they do not require activation to kill cells. T cells play a major role in cell-mediated immunity (not involving antibodies). Their T cell receptors (TCRs) differentiate from other lymphocyte types. The thymus, a specialized organ of the immune system, is primarily responsible for the maturation of T cells. There are six types of T cells: helper T cells (e.g., CD4+ cells), cytotoxic T cells (TCs, also known as cytotoxic T lymphocytes, CTLs, T killer cells, cytolytic T cells, CD8+ T cells, or killer T cells), memory T cells ((i) stem memory TSCM cells are CD45RO-, CCR7+, CD45RA+, CD62L+ (L-selectin), CD27+, CD28+, and IL-7Rα+, like naive cells, but also express large amounts of CD95, IL-2Rβ, CXCR3, and LFA-1, making them memory cells. (ii) central memory T cells express L-selectin and CCR7 and secrete IL-2 but not IFNγ or IL-4, however (iii) effector memory T cells do not express L-selectin or CCR7 but produce effector cytokines such as IFNγ and IL-4), regulatory T cells (Treg, suppressor T cells, or CD4+CD25+ regulatory T cells), natural killer T cells (NKT), and gamma delta T cells. On the other hand, B cells play a major role in humoral immunity (involving antibodies). B cells produce antibodies and antigens, act as antigen-presenting cells (APCs), and turn into memory B cells after activation by antigen interaction. In mammals, immature B cells are formed in the bone marrow, from which the name originates.

In the context of this disclosure, the terms "TN", "T naive-like", and CCR7+CD45RA+ refer to cells that are actually closer to stem cell-like memory cells than to regular naive T cells. Thus, all references to _TN in the examples and claims refer to cells that have been experimentally selected solely by their characterization as CCR7+CD45RA+ cells and should be interpreted as such. Although their more preferred name in the context of this disclosure is stem cell-like memory cells, they are referred to as CCR7+CD45RA+ cells. Further characterization of stem cell-like memory cells is described, for example, in Arihara Y, Jacobsen CA, Armand P, et al. Journal for ImmunoTherapy of Cancer. 2019;7(1):P210.

The term "genetically engineered" or "engineered" refers to a method of modifying the genome of a cell, including, but not limited to, deleting a coding or non-coding region or portion thereof, or inserting a coding region or portion thereof. In some embodiments, the cell being modified is a lymphocyte, e.g., a T cell, which may be obtained from either a patient or a donor. The cell may be modified to express an exogenous construct, such as, for example, a chimeric antigen receptor (CAR) or a T cell receptor (TCR), and the exogenous construct is integrated into the genome of the cell.

"Immune response" refers to the action of cells of the immune system (e.g., T lymphocytes, B lymphocytes, natural killer (NK) cells, macrophages, eosinophils, mast cells, dendritic cells, and neutrophils) and soluble macromolecules (including Abs, cytokines, and complement) produced by either these cells or the liver that result in the selective targeting, binding, damaging, destroying, and/or elimination from the vertebrate body of invading pathogens, pathogen-infected cells or tissues, cancerous or other abnormal cells, or, in the case of autoimmunity or pathological inflammation, normal human cells or tissues.

The term "immunotherapy" refers to the treatment of a subject suffering from a disease or at risk of suffering from or relapsing from a disease by methods that include inducing, enhancing, suppressing, or otherwise modifying an immune response. Examples of immunotherapy include, but are not limited to, T cell therapy. T cell therapy may include adoptive T cell therapy, tumor infiltrating lymphocyte (TIL) immunotherapy, autologous cell therapy, genetically engineered autologous cell therapy (eACT™), and allogeneic T cell transplantation. However, one of skill in the art will appreciate that the pretreatment methods disclosed herein enhance the efficacy of any transplanted T cell therapy. Examples of T cell therapy are described in U.S. Patent Application Publication Nos. 2014/0154228 and 2002/0006409, U.S. Patent No. 7,741,465, U.S. Patent No. 6,319,494, U.S. Patent No. 5,728,388, and WO 2008/081035. In some embodiments, the immunotherapy comprises a CAR T cell therapy. In some embodiments, the CAR T cell therapy product is administered by injection.

T cells for immunotherapy may be derived from any source known in the art. For example, T cells may be differentiated in vitro from a hematopoietic stem cell population or may be obtained from a subject. T cells may be obtained, for example, from peripheral blood mononuclear cells (PBMCs), bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, splenic tissue, and tumors. Additionally, T cells may be derived from one or more T cell lines available in the art. T cells may also be obtained from a unit of blood drawn from a subject using a variety of techniques known to those of skill in the art, such as FICOLL™ separation and/or apheresis. Additional methods of isolating T cells for T cell therapy are disclosed in U.S. Patent Application Publication No. 2013/0287748, which is incorporated herein by reference in its entirety.

The term "genetically engineered autologous cell therapy" or "eACT™," also known as adoptive cell transfer, is a process in which a patient's own T cells are harvested and then genetically modified to recognize and target one or more antigens expressed on the cell surface of one or more specific tumor cells or malignancies. The T cells can be engineered, for example, to express a chimeric antigen receptor (CAR). CAR-positive (+) T cells are engineered to express an extracellular single-chain variable fragment (scFv) specific for a particular tumor antigen linked to an intracellular signaling moiety that includes at least one costimulatory domain and at least one activation domain. CAR scFvs can be designed to target CD19, a transmembrane protein expressed by cells of the B cell lineage, including, for example, all normal B cells, and B cell malignancies, including, but not limited to, diffuse large B cell lymphoma (DLBCL)-unspecified type, primary mediastinal large B cell lymphoma, high-grade B cell lymphoma, and DLBCL arising from follicular lymphoma, NHL, CLL, and non-T cell ALL. Exemplary CAR T cell therapies and constructs are described in U.S. Patent Application Publication Nos. 2013/0287748, 2014/0227237, 2014/0099309, and 2014/0050708, which references are incorporated by reference in their entireties.

As used herein, a "patient" or "subject" includes any human suffering from cancer (e.g., lymphoma or leukemia). The terms "subject" and "patient" are used interchangeably herein.

As used herein, the term "in vitro cells" refers to any cells cultured ex vivo. In particular, in vitro cells can include T cells. The term "in vivo" means within a patient.

The terms "peptide," "polypeptide," and "protein" are used interchangeably and refer to compounds composed of amino acid residues covalently linked by peptide bonds. A protein or peptide contains at least two amino acids, with no limit placed on the maximum number of amino acids that may make up a protein or peptide sequence. A polypeptide includes any peptide or protein that contains two or more amino acids linked together by peptide bonds. As used herein, the term refers to both short chains, also commonly referred to in the art as peptides, oligopeptides, and oligomers, for example, and longer chains, generally referred to in the art as proteins, of which there are many varieties. "Polypeptides" include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, among others. A polypeptide includes natural peptides, recombinant peptides, synthetic peptides, or combinations thereof.

As used herein, "stimulation" refers to a primary response elicited by binding of a stimulatory molecule to its cognate ligand, where the binding mediates a signaling event. A "stimulatory molecule" is a molecule on a T cell, e.g., a T cell receptor (TCR)/CD3 complex that specifically binds to a cognate stimulatory ligand present on an antigen presenting cell. A "stimulatory ligand" is a ligand that, when present on an antigen presenting cell (e.g., APC, dendritic cell, B cell, etc.), specifically binds to a stimulatory molecule on a T cell, thereby mediating a primary response by the T cell, including, but not limited to, activation, initiation of an immune response, proliferation, etc. Stimulatory ligands include, but are not limited to, anti-CD3 antibodies, peptide-loaded MHC class I molecules, superagonist anti-CD2 antibodies, and superagonist anti-CD28 antibodies.

As used herein, a "costimulatory signal" refers to a signal that, in combination with a primary signal, such as TCR/CD3 ligation, triggers a T cell response, including, but not limited to, proliferation and/or upregulation or downregulation of key molecules.

As used herein, a "costimulatory ligand" includes a molecule on an antigen-presenting cell that specifically binds to a cognate costimulatory molecule on a T cell. Binding of a costimulatory ligand provides a signal that mediates a T cell response, such as, but not limited to, proliferation, activation, and differentiation. A costimulatory ligand induces a signal, for example, by binding of the T cell receptor (TCR)/CD3 complex to a peptide-loaded major histocompatibility complex (MHC) molecule, in addition to the primary signal provided by the stimulatory molecule. Costimulatory ligands may include, but are not limited to, 3/TR6, 4-1BB ligand, an agonist or antibody that binds to the Toll ligand receptor, B7-1 (CD80), B7-2 (CD86), CD30 ligand, CD40, CD7, CD70, CD83, herpes virus entry mediator (HVEM), human leukocyte antigen G (HLA-G), ILT4, immunoglobulin-like transcript (ILT)3, inducible costimulatory ligand (ICOS-L), intercellular adhesion molecule (ICAM), a ligand that specifically binds to B7-H3, lymphotoxin beta receptor, MHC class I chain-related protein A (MICA), MHC class I chain-related protein B (MICB), OX40 ligand, PD-L2, or programmed cell death (PD) L1. In certain embodiments, the costimulatory ligand includes, but is not limited to, a costimulatory molecule present on a T cell, such as, but not limited to, a ligand that specifically binds to 4-1BB, B7-H3, CD2, CD27, CD28, CD30, CD40, CD7, ICOS, CD83, lymphocyte function-associated antigen-1 (LFA-1), natural killer cell receptor C (NKG2C), OX40, PD-1, or an antibody that specifically binds to tumor necrosis factor superfamily member 14 (TNFSF14 or LIGHT).

A "costimulatory molecule" is a cognate binding partner on a T cell that specifically binds to a costimulatory ligand, thereby mediating a costimulatory response by the T cell, such as, but not limited to, proliferation. Costimulatory molecules include, but are not limited to, 4-1BB/CD137, B7-H3, BAFFR, BLAME (SLAMF8), BTLA, CD33, CD45, CD100 (SEMA4D), CD103, CD134, CD137, CD154, CD16, CD160 (BY55), CD18, CD19, CD19a, CD2, CD22, CD247, CD27, CD276 (B7-H3), CD2 8, CD29, CD3 (α; beta, delta, epsilon, gamma, ζ), CD30, CD37, CD4, CD4, CD40, CD49a, CD49D, CD49f, CD5, CD64, CD69, CD7, CD80, CD83 ligand, CD84, CD86, CD8α, CD8β, CD9, CD96 (Tactile), CD11a, CD11b, CD11c, CD11d, CDS, CEACAM1, CRT AM, DAP-10, DNAM1 (CD226), Fcγ receptor, GADS, GITR, HVEM (LIGHTR), IA4, ICAM-1, ICOS, Igα (CD79a), IL2Rβ, IL2Rγ, IL7Rα, integrin, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB2, ITGB7, ITGB1, KIRDS2, LAT, LFA-1, LIGHT (tumor necrosis factor superfamily member 14; TNFSF14), LTBR, Ly9 (CD229), lymphocyte function-associated antigen-1 (LFA-1 (CD11a/CD18), M HC class I molecules, NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80 (KLRF1), OX40, PAG/Cbp, PD-1, PSGL1, SELPLG (CD162), signaling lymphocyte activation molecule, SLAM (SLAMF1; CD150; IPO-3), SLAMF4 (CD244; 2B4), SLAMF6 (NTB-A, Ly108), SLAMF7, SLP-76, TNF, TNFr, TNFR2, Toll ligand receptor, TRANCE/RANKL, VLA1, or VLA-6, or fragments, truncations, or combinations thereof.

The terms "reduce" and "decrease" are used interchangeably herein and refer to any change that is less than the original. "Reduce" and "reduce" are relative terms and require a comparison between measurements before and after. "Reduce" and "reduce" encompass complete depletion. Similarly, the term "increase" refers to any change that is higher than the original value. "Increase", "higher", and "lower" are relative terms and require a comparison between measurements before and after and/or a comparison between a reference standard. In some embodiments, the reference value is obtained from a general population, which may be the general population of patients. In some embodiments, the reference value is derived from a quartile analysis of the general patient population.

"Treatment" or "treating" of a subject refers to any type of intervention or process performed on a subject, or administration of an active agent to a subject, for the purpose of reversing, alleviating, ameliorating, inhibiting, delaying, or preventing the onset, progression, development, severity, or recurrence of a symptom, complication, or condition, or biochemical manifestations associated with a disease. In some embodiments, "treatment" or "treating" encompasses partial remission. In another embodiment, "treatment" or "treating" includes complete remission. In some embodiments, treatment may be prophylactic, in which case the treatment is administered before any symptoms of the condition are observed. As used herein, the term "prophylaxis" refers to the prevention of or protective treatment against a disease or condition. Prevention of a symptom, disease, or condition may include, for example, a reduction (e.g., alleviation) of one or more symptoms of the disease or condition compared to a reference level (e.g., symptoms in a similar subject not administered the treatment). Prevention can also include, for example, delaying the onset of one or more symptoms of a disease or condition, as compared to a reference level (e.g., the onset of a symptom in a similar subject not receiving treatment). In embodiments, the disease is a disease described herein. In some embodiments, the disease is cancer. In some embodiments, the pathological condition is CRS or neurotoxicity. In some embodiments, indicators of improvement or successful treatment include a determination of no score on a toxicity rating scale (e.g., a CRS or neurotoxicity rating scale), e.g., a score of less than 3, or a change in grade or severity on a rating scale described herein, e.g., a change from a score of 4 to a score of 3, or a change from a score of 4 to a score of 2, 1, or 0.

As used herein, the term "polyfunctional T cells" refers to cells that co-secrete at least two proteins from a pre-specified panel per cell in combination with the amount of each protein produced (i.e., a combination of the number of secreted proteins and their intensity). In some embodiments, a single cell functional profile is determined for each evaluable population of engineered T cells. The profiles can be classified into effector (granzyme B, IFN-γ, MIP-1α, perforin, TNF-α, TNF-β), stimulatory (GM-CSF, IL-2, IL-5, IL-7, IL-8, IL-9, IL-12, IL-15, IL-21), regulatory (IL-4, IL-10, IL-13, IL-22, TGF-beta1, sCD137, sCD40L), chemoattractant (CCL-11, IP-10, MIP-1β, RANTES), and inflammatory (IL-1 b, IL-6, IL-17 A, IL-17 F, MCP-1, MCP-4) groups. In some embodiments, the functional profile of each cell allows the calculation of other metrics, including breakdown of each sample by cell multifunctionality (i.e., what percentage of cells secrete multiple cytokines compared to non-secreting or monofunctional cells) and breakdown of samples by functional groups (i.e., which mono- and polyfunctional groups are secreted by cells in the sample and their frequency).

As used herein, "myeloid cells" are a subpopulation of white blood cells that include granulocytes, monocytes, macrophages, and dendritic cells.

As used herein, the term "quartiles" is a statistical term that describes the division of observations into four predefined intervals based on the values of the data and their difference from the full set of observations.

As used herein, the term "Study Day 0" is defined as the day a subject first receives a CAR T cell infusion. The day prior to Study Day 0 is Study Day -1. Any day after enrollment and prior to Study Day -1 is consecutive and a negative integer value.

As used herein, the term "durable response" refers to subjects with a sustained response at least one year of follow-up after CAR T cell infusion. In one embodiment, "duration of response" (DOR) is defined only for subjects who experience an objective response and is the time from first objective response to disease progression (per Cheson et al., 2014) or disease-related death, whichever occurs first.

As used herein, the term "relapse" refers to a subject who achieves a complete response (CR) or partial response (PR) and then experiences disease progression.

As used herein, the term "non-responder" refers to a subject who has not experienced a CR or PR following CAR T cell infusion.

As used herein, the term "objective response" refers to complete response (CR), partial response (PR), or failure, as defined by the IWG Response Criteria for Malignant Lymphoma (Cheson et al., J Clin Oncol. 2007;25(5):579-86).

As used herein, the term "complete response" refers to a complete reversal of disease that becomes undetectable by radioimaging and clinical laboratory assessment; there is no sign of cancer at a given time point.

As used herein, the term "partial response" refers to a greater than 30% reduction in tumor size without complete recovery.

As used herein, "objective response rate" (ORR) is determined according to the International Working Group (IWG) 2007 criteria (Cheson et al. J Clin Oncol. 2007;25(5):579-86).

As used herein, "progression-free survival (PFS)" may be defined as the time from the date of T cell infusion to the date of disease progression or death from any cause. Progression is defined according to investigator assessment of response as defined by the IWG criteria (Cheson et al., J Clin Oncol. 2007;25(5):579-86).

The term "overall survival (OS)" may be defined as the time from the date of T cell infusion to the date of death from any cause.

As used herein, the proliferation and persistence of CAR T cells in peripheral blood can be monitored by qPCR analysis using CAR-specific primers for the scFv portion of the CAR (e.g., the heavy chain of the CD19 binding domain) and its hinge/CD28 transmembrane domain. Alternatively, it can be measured by counting CAR cells/blood unit volume.

As used herein, blood collection schedules for CAR T cells can be prior to CAR T cell infusion, 7 days, 2 weeks (day 14), 4 weeks (day 28), 3 months (day 90), 6 months (day 180), 12 months (day 360), and 24 months (day 720).

As used herein, "peak CAR T cells" is defined as the maximum absolute number of CAR+ PBMCs/μL in serum achieved after day 0.

As used herein, "time to peak CAR T cells" is defined as the number of days from day 0 to the day that peak CAR T cells are achieved.

As used herein, "Area Under Curve (AUC) of CAR T cell levels from day 0 to day 28" is defined as the area under the curve in a plot of CAR T cell levels versus scheduled visits from day 0 to day 28. The AUC is the total level of CAR T cells over time.

As used herein, blood collection schedules for cytokines are the day before or the day of conditioning chemotherapy (day -5), days 0, 1, 3, 5, 7, every other day during hospitalization if hospitalized, week 2 (day 14), and week 4 (day 28).

As used herein, a "baseline" cytokine is defined as the last value measured prior to conditioning chemotherapy.

As used herein, the fold change from baseline on day X is

として定義される。

It is defined as:

As used herein, "post-baseline cytokine peak" is defined as the maximum serum cytokine level achieved by day 28 after baseline (day -5).

As used herein, "time to peak cytokine" following CAR T cell infusion is defined as the number of days from day 0 to the day peak cytokine is achieved.

As used herein, the "area under the curve (AUC) of cytokine levels" from day -5 to day 28 is defined as the area under the curve in a plot of cytokine levels versus scheduled visits from day -5 to day 28. This AUC is the total level of cytokine over time. Assuming that cytokines and CAR+ T cells are measured at certain discrete time points, the trapezoidal rule can be used to estimate the AUC.

As used herein, a technical emergency adverse event (TEAE) is defined as an adverse event (AE) occurring at or after the first dose of conditioning chemotherapy. Adverse events may be coded using the MedDRA version 22.0 and graded using the National Cancer Institute's (NCI) Common Terminology Criteria for Adverse Events (CTCAE) version 4.03. Cytokine release syndrome (CRS) events may be graded at the syndrome level according to Lee et al. (Lee et al, 2014 Blood. 2014;124(2):188-95). Individual CRS symptoms may be graded according to CTCAE 4.03. Neurological events may be graded according to, for example, Topp, MS et al. Lancet Oncology. These can be identified using a discovery strategy based on known neurotoxicity associated with CAR T immunotherapy, as described in 2015;16(1):57-66.

Various aspects of the present disclosure are described in further detail in the following subsections.

Pre-Treatment Attributes Pre-treatment characteristics of apheresis cells and immune cells (also referred to as engineered cells such as T cells), as well as patient immunological factors measured from patient samples, can be used to assess the probability of clinical outcome, including response and toxicity. Characteristics associated with clinical outcome can be tumor-related parameters (e.g., tumor burden, serum LDH as a marker of hypoxia/cell death, inflammatory markers associated with tumor burden and myeloid cell activity), T cell characteristics (e.g., T cell compatibility, function, particularly IFNγ production associated with T1, and total number of infused CD8 T cells), and CAR T cell engraftment as measured by peak CAR T cell levels in the blood at early time points.

Information inferred from the T cell characteristics and the patient's pre-treatment characteristics can be used to determine, refine, or adjust a therapeutically effective dose suitable for treating a malignancy (e.g., cancer). Additionally, some T cell characteristics and the patient's pre-treatment characteristics can be used to determine whether a patient will develop an adverse event (e.g., neurotoxicity (NT), cytokine release syndrome (CRS)) following treatment with engineered chimeric antigen receptor (CAR) immunotherapy. As a result, an effective adverse event management strategy (e.g., administration of tocilizumab, corticosteroid treatment, or anti-seizure drugs to prevent toxicity based on the measured levels of one or more characteristics) can be determined.

In some embodiments, the pre-treatment characteristics are characteristics of engineered T cells that contain one or more chimeric antigen receptors. In some embodiments, the pre-treatment characteristics are T cell transduction rate, predominant T cell phenotype, number of CAR T cells and T cell subsets, CAR T cell compatibility, T cell functionality, T cell polyfunctionality, number of differentiated CAR+CD8+ T cells, number of CCR7+CD45RA+ T cells, CD4/CD8 ratio, IFN-γ in culture).

In some embodiments, the pre-treatment characteristic is measured from a sample obtained from the patient (e.g., cerebrospinal fluid (CSF), blood, serum, or tissue biopsy). In some embodiments, the one or more pre-treatment characteristics is tumor burden, IL-6 levels, or LDH levels.

T Cell Compatibility In some embodiments, intrinsic cell compatibility is assessed based on the ability of CAR T cells to expand during non-specific stimulation in vitro (e.g., shorter doubling time), the differentiation state of the CAR T cells (favorable juvenile phenotype), the level of specialized CAR T cell subsets in the CAR T cell population (e.g., the number of CD8 and naive-like CD8 cells (e.g., CD8+CCR7+CD45RA+ T cells) in the infusion product), and in vivo CAR T cell expansion rates.

In one embodiment, T cell compatibility is the ability of the cells to proliferate rapidly. In the context of engineered T cells, in one embodiment, T cell compatibility is a measure of how quickly an engineered T cell population proliferates prior to treatment. As described herein, T cell compatibility is an attribute of engineered T cells that correlates with clinical outcome. In some embodiments, T cell compatibility is measured by doubling time or proliferation rate. An exemplary derivation of T cell "compatibility" measured as a T cell population doubling time (DT) during the manufacturing process is provided below:

Duration may be defined as the total manufacturing time frame minus 3 days (essentially the number of days of produced cells in culture after transduction and before harvesting and cryopreservation). Recombinant IL-2 (following non-specific stimulation with, for example, anti-CD3 antibodies) can be used to drive polyclonal T cell expansion towards achieving the target dose. The shorter the DT, the more compatible the engineered T cells are. In vitro expansion rate can be calculated using the following formula:
Growth rate = ln(2)/doubling time In the above example, the growth rate is provided in units of "rate/day" or "/day".

In some embodiments, the in vivo proliferation rate is measured by enumerating CAR cells/unit of blood volume. In some embodiments, the in vivo proliferation rate is measured by CAR gene copy number per μg of host DNA. In some embodiments, the in vivo proliferation rate is measured by enumerating CAR cells/unit of blood volume.

As described herein, higher peak proliferation of CAR T cells in peripheral blood, which generally occurs within 2 weeks after CAR T cell infusion, can be associated with both objective and sustained responses, defined as sustained response with a minimum follow-up of 1 year. Peak number of CAR T cells in blood correlated with response. Cumulative CAR T cell levels over the first 28 days, as measured by area under the curve (AUC) in blood, can also be associated with better objective and sustained responses to treatment. In some embodiments, CAR T cell levels are calculated by counting the number of CAR T cells per unit of blood volume. In one embodiment, higher peak proliferation of CAR T cells in peripheral blood refers to peak proliferation values that fall within a higher quartile. In some embodiments, in vivo proliferation rate is measured by enumerating CAR cells/unit of blood volume. In some embodiments, in vivo proliferation rate is measured by CAR gene copy number per μg of host DNA.

As described herein, the inherent ability of pretreatment to measure T cell proliferation as measured by product doubling time is a key attribute of product T cell compatibility. Compared to other product characteristics, DT was most strongly associated with the frequency of T cell differentiation subsets in the final infusion bag. Specifically, DT was positively associated with the frequency of effector memory T (TEM) cells and negatively associated with the frequency of naive-like T (TN) cells. In one embodiment (e.g., axicabtageneciloleucel), _TN cells identified as CCR7+CD45RA+ cells are in fact stem cell-like memory cells and not regular naive T cells. As described herein, baseline tumor burden is positively associated with differentiation phenotype in the final infusion product. As described herein, product composition and clinical outcomes are related to the patient's pretreatment immune status. Thus, in one embodiment, the present disclosure provides a method of reducing tumor burden after treatment with CAR T cells, comprising administering an infusion product with an increased frequency of naive-like T (TN) cells in the infusion product compared to baseline. In another embodiment, the disclosure provides a method of predicting or estimating a differentiation phenotype of a final infusion product, comprising measuring a patient's baseline tumor burden to obtain a value, and estimating or predicting the differentiation phenotype based on the value. In one embodiment, the measure further comprises adjusting an effective dose of CAR T cells in the final product based on the value.

T Cell Phenotype As described herein, the T cell phenotype at the time of manufacturing the starting material (apheresis) may be related to T cell compatibility (DT). The combined % of Tn-like and Tcm cells (CCR7+ cells) inversely correlates with DT. The % of Tem (CCR7-CD45RA-) cells is directly related to DT. Thus, in some embodiments, the pre-treatment profile is the % of Tn-like and Tcm cells. In some embodiments, the % of Tn-like and Tcm cells is determined by the percentage of CCR7+ cells. In some embodiments, the percentage of CCR7+ cells is measured by flow cytometry.

In some embodiments, the pre-treatment characteristic is % Tem (CCR7-CD45RA-) cells. In some embodiments, the % Tem cells is determined by the percentage of CCR7-CD45RA- cells. In some embodiments, the percentage of CCR7-CD45RA- cells is measured by flow cytometry.

As described herein, the higher the percentage of total CD3+ T cells or effector memory T cells within the CD4 and CD8 subsets in the apheresis product, the longer the manufacturing doubling time. However, as described herein, the more immature the T cell phenotype in the starting material, the better the product T cell compatibility. As described herein, CD27+CD28+ T _N cells expressing costimulatory molecules, which represent an immune-competent subset of T _N cells, show a positive association with product doubling time. As described herein, there is a direct association across all major phenotypic groups, including the proportion of T cell subsets defined by differentiation markers in the CD3, CD4, and CD8 subpopulations in the apheresis product compared to the final product phenotype. As described herein, the proportion of T cells with CD25 ^hi CD4 expression in the apheresis material, likely representing regulatory T cells, shows a negative correlation with the CD8 T cell output in the product. As described herein, tumor burden after CAR T cell therapy shows a positive association with the differentiation phenotype of the final product.

T1 Function The engineered T cells may be characterized by their immune function characteristics. The methods of the present disclosure provide for the measurement of ex vivo cytokine production levels. In some embodiments, the cytokines are selected from the group consisting of IFNγ, TNFa, IL-12, MIP1β, MIP1α, IL-2, IL-4, IL-5, and IL-13. In some embodiments, T cell function is measured by the levels of Th1 cytokines.

In some embodiments, the Th1 cytokine is selected from the group consisting of IFNγ, TNFa, and IL-12. In some embodiments, T cell function is measured by the level of IFNγ production. In some embodiments, excess T cell IFNγ (pre-treatment signature) and T1 activity after treatment are signatures that can be used to determine if a patient will develop an adverse event (e.g., neurotoxicity). In some embodiments, the level of IFNγ produced by the engineered CAR T cells is measured by co-culture prior to administration of the engineered CAR T cells.

Other Immune Cell Product Characteristics In some embodiments, the immune cell product administered to a subject has several other product characteristics related to its efficacy. In some embodiments, the product characteristics are selected from: total number of CAR T cells per μL, total number of T cells per μL, transduction rate, %, IFN-γ level, pg/mL, viability, %, CD4/CD8 ratio; naive (CCR7+CD45RA+) T cells; %, central memory (CCR7+CD45RA-) T cells; %, ( _TN + _TCM )/( _TEM + _TEFF ) ratio ( _TCM , central memory T cells; _TEFF , effector T cells; _TEM , effector memory T cells; _TN , naive T cells).

Chimeric Antigen Receptors Chimeric antigen receptors (CARs) are engineered receptors. These engineered receptors can be inserted into and expressed in immune cells, including T cells and other lymphocytes, according to techniques known in the art. With CARs, a single receptor can be programmed to both recognize a specific antigen and, when bound to that antigen, activate the immune cell to attack and destroy cells bearing that antigen. If these antigens are present on tumor cells, immune cells expressing the CAR can target and kill the tumor cells. Chimeric antigen receptors can incorporate costimulatory (signaling) domains to enhance their efficacy. See U.S. Patent Nos. 7,741,465 and 6,319,494, as well as Krause et al. and Finney et al. (supra), Song et al., Blood 119:696-706 (2012); Kalos et al., J. Immunol. 199: 113-116 (2012); , Sci. Transl. Med. 3:95 (2011); Porter et al., N. Engl. J. Med. 365:725-33 (2011); and Gross et al., Annu. Rev. Pharmacol. Toxicol. 56:59-83 (2016).

In some embodiments, the costimulatory domain comprising a truncated hinge domain ("THD") further comprises some or all of a member of the immunoglobulin family, e.g., IgG1, IgG2, IgG3, IgG4, IgA, IgD, IgE, IgM, or a fragment thereof.

In some embodiments, the THD is derived from a human complete hinge domain ("CHD"). In other embodiments, the THD is derived from a CHD of a rodent, murine, or primate (e.g., non-human primate) costimulatory protein. In some embodiments, the THD is derived from a chimeric CHD of a costimulatory protein.

The costimulatory domain of the CAR of the present disclosure may further comprise a transmembrane domain and/or an intracellular signaling domain. The transmembrane domain may be fused to the extracellular domain of the CAR. The costimulatory domain may similarly be fused to the intracellular domain of the CAR. In some embodiments, a transmembrane domain that is naturally associated with one of the domains in the CAR is used. In some cases, the transmembrane domain is selected or modified by amino acid substitution to avoid association with the transmembrane domain of the same or different surface membrane protein as such domain, minimizing interaction with other members of the receptor complex. The transmembrane domain may be derived from either natural or synthetic sources. If of natural origin, the domain may be derived from any membrane-bound or transmembrane protein. Transmembrane regions that are particularly useful in the present disclosure include 4-1BB/CD137, activating NK cell receptor, immunoglobulin proteins, B7-H3, BAFFR, BLAME (SLAMF8), BTLA, CD100 (SEMA4D), CD103, CD160 (BY55), CD18, CD19, CD19a, CD2, CD247, CD27, CD276 (B7-H3), CD28, CD29, CD3δ, CD3ε, CD3γ, CD3ζ, CD30, CD4, CD40, CD49a, CD49D, CD49f, CD69, CD7, CD84, CD8, CD8α, CD8β, CD96 (Tactile), CD11a, CD11b, CD11c, CD11d, CDS, CEACAM1, CRT1, CRT2, CRT3, CRT4, CRT5, CRT6, CRT7, CRT8, CRT9, CRT10, CRT11, CRT12, CRT13, CRT14, CRT15, CRT16, CRT17, CRT18, CRT19, CRT20, CRT21, CRT22, CRT23, CRT24, CRT25, CRT26, CRT27, CRT30, CRT31, CRT32, CRT33, CRT34, CRT35, CRT36, CRT37, CRT38, CRT40, CRT49, CRT51, CRT42, CRT43, CRT44, CRT45, CRT46, CRT47, CRT52, CRT53, CRT54, CRT55, CRT56, CRT57, CRT58, CRT59, CRT60, CRT61, CRT62, CRT63, CRT64 AM, cytokine receptor, DAP-10, DNAM1 (CD226), Fcγ receptor, GADS, GITR, HVEM (LIGHTR), IA4, ICAM-1, Igα (CD79a), IL-2Rβ, IL-2Rγ, IL-7Rα, inducible T cell costimulatory molecule (ICOS), integrin, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB2, ITGB7, ITGB1, KIRDS2, LAT, LFA-1, ligand specifically binding to CD83, LIGHT, LTBR, Ly9 (CD229), lymphocyte function-associated antigen-1 (LFA-1; CD11a/CD18), MHC class I molecule, NKG2C, NKG2D, NK p30, NKp44, NKp46, NKp80 (KLRF1), OX-40, PAG/Cbp, programmed cell death 1 (PD-1), PSGL1, SELPLG (CD162), signaling lymphocyte activation molecule (SLAM protein), SLAM (SLAMF1; CD150; IPO-3), SLAMF4 (CD244; 2B4), SLAMF6 (NTB-A, Lyl08), SLAMF7, SLP-76, TNF receptor protein, TNFR2, TNFSF14, Toll ligand receptor, TRANCE/RANKL, VLA1, or VLA-6, or a fragment, truncation, or combination thereof (e.g., may include at least the transmembrane domain).

Optionally, a short linker may form the bond between any or some of the extracellular, transmembrane, and intracellular domains of the CAR. In some embodiments, the linker may be derived from glycine-glycine-glycine-glycine-serine repeats (SEQ ID NO:2, (G4S)n) or GSTSGSGKPGSGEGSTKG (SEQ ID NO:1). In some embodiments, the linker comprises 3-20 amino acids and comprises an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to GSTSGSGKPGSGEGSTKG (SEQ ID NO:1).

The linkers described herein may be used as peptide tags. The linker peptide sequence may be of any length suitable for linking one or more proteins of interest, and is preferably designed to be sufficiently flexible to allow for proper folding and/or function and/or activity of one or both of the peptides it links. Thus, the linker peptide may have a length of 10 amino acids or less, 11 amino acids or less, 12 amino acids or less, 13 amino acids or less, 14 amino acids or less, 15 amino acids or less, 16 amino acids or less, 17 amino acids or less, 18 amino acids or less, 19 amino acids or less, or 20 amino acids or less. In some embodiments, the linker peptide is at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 amino acids in length. In some embodiments, the linker comprises at least 7 amino acids and no more than 20 amino acids, at least 7 amino acids and no more than 19 amino acids, at least 7 amino acids and no more than 18 amino acids, at least 7 amino acids and no more than 17 amino acids, at least 7 amino acids and no more than 16 amino acids, at least 7 amino acids and no more than 15 amino acids, at least 7 amino acids and no more than 14 amino acids, at least 7 amino acids and no more than 13 amino acids, at least 7 amino acids and no more than 12 amino acids, or at least 7 amino acids and no more than 11 amino acids. In certain embodiments, the linker comprises 15-17 amino acids, and in certain embodiments, 16 amino acids. In some embodiments, the linker comprises 10-20 amino acids. In some embodiments, the linker comprises 14-19 amino acids. In some embodiments, the linker comprises 15-17 amino acids. In some embodiments, the linker comprises 15-16 amino acids. In some embodiments, the linker comprises 16 amino acids. In some embodiments, the linker comprises 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids.

In some embodiments, a spacer domain is used. In some embodiments, the spacer domain is derived from CD4, CD8a, CD8b, CD28, CD28T, 4-1BB, or other molecules described herein. In some embodiments, the spacer domain may include a chemically inducible dimerizer to control expression by addition of small molecules. In some embodiments, a spacer is not used.

The intracellular (signaling) domain of the engineered T cells of the present disclosure can provide signaling to the activation domain, which then activates at least one of the normal effector functions of an immune cell. The effector function of a T cell can be, for example, cytolytic activity or helper activity, including secretion of cytokines.

In certain embodiments, suitable intracellular signaling domains include, but are not limited to, 4-1BB/CD137, activating NK cell receptor, immunoglobulin proteins, B7-H3, BAFFR, BLAME (SLAMF8), BTLA, CD100 (SEMA4D), CD103, CD160 (BY55), CD18, CD19, CD19a, CD2, CD247, CD27, CD276 (B7-H3), CD28, CD29, CD3δ, CD3ε, CD3γ, CD30, CD4, CD40, CD49a, CD49D, CD49f, CD69, CD7, CD84, CD8, CD8α, CD8β, CD96 (Tactile), CD11a, CD11b, CD11c, CD11d, CDS, CEACAM1, CRT AM, cytokine receptor, DAP-10, DNAM1 (CD226), Fcγ receptor, GADS, GITR, HVEM (LIGHTR), IA4, ICAM-1, Igα (CD79a), IL-2Rβ, IL-2Rγ, IL-7Rα, inducible T cell costimulatory molecule (ICOS), integrin, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB2, ITGB7, ITGB1, KIRDS2, LAT, ligand that specifically binds to CD83, LIGHT, LTBR, Ly9 (CD229), Ly108), lymphocyte function-associated antigen-1 (LFA-1; CD11a/CD18), MHC classifier NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80 (KLRF1), OX-40, PAG/Cbp, programmed cell death 1 (PD-1), PSGL1, SELPLG (CD162), signaling lymphocyte activation molecule (SLAM protein), SLAM (SLAMF1; CD150; IPO-3), SLAMF4 (CD244; 2B4), SLAMF6 (NTB-A, SLAMF7, SLP-76, TNF receptor protein, TNFR2, TNFSF14, Toll ligand receptor, TRANCE/RANKL, VLA1, or VLA-6, or fragments, truncations, or combinations thereof.

Antigen Binding Molecules Suitable CARs may bind antigens (such as cell surface antigens) by incorporating an antigen binding molecule that interacts with its target antigen. In some embodiments, the antigen binding molecule is an antibody fragment thereof, such as one or more single chain antibody fragments ("scFv"). An scFv is a single chain antibody fragment having the variable regions of the antibody heavy and light chains linked together. See U.S. Patent Nos. 7,741,465 and 6,319,494, and Eshhar et al., Cancer Immunol Immunotherapy (1997) 45:131-136. ScFvs retain the ability of the parent antibody to specifically interact with the target antigen. ScFvs are useful for chimeric antigen receptors because they can be engineered to be expressed as part of a single chain along with other CAR components (Id.). Krause et al., J. Exp. Med. , Volume 188, No. 4, 1998(619-626); Finney et al., Journal of Immunology, 1998, 161:2791-2797. It will be understood that an antigen binding molecule is typically contained within the extracellular portion of the CAR such that it is capable of recognizing and binding to an antigen of interest. Bispecific and multispecific CARs are contemplated within the scope of the present disclosure, having specificity for two or more targets of interest.

In some embodiments, the polynucleotide encodes a CAR comprising a (truncated) hinge domain and an antigen binding molecule that specifically binds to a target antigen. In some embodiments, the target antigen is a tumor antigen. In some embodiments, the antigen is a tumor associated surface antigen, e.g., 5T4, alpha fetoprotein (AFP), B7-1 (CD80), B7-2 (CD86), BCMA, B-human chorionic gonadotropin, CA-125, carcinoembryonic antigen (CEA), CD123, CD133, CD138, CD19, CD20, CD22, CD23, CD24, CD25, CD30, CD33, CD34, CD4, CD40, CD44, CD56, CD8 , CLL-1, c-Met, CMV-specific antigen, CS-1, CSPG4, CTLA-4, DLL3, disialoganglioside GD2, ductal epithelial mucin, EBV-specific antigen, EGFR variant III (EGFRvIII), ELF2M, endoglin, ephrinB2, epidermal growth factor receptor (EGFR), epithelial cell adhesion molecule (EpCAM), epithelial tumor antigen, ErbB2 (HER2/neu), fibroblast-associated protein (fap), FLT3, folate Binding proteins, GD2, GD3, glioma-associated antigens, glycosphingolipids, gp36, HBV-specific antigens, HCV-specific antigens, HER1-HER2, HER2-HER3 combinations, HERV-K, high molecular weight melanoma-associated antigen (HMW-MAA), HIV-1 envelope glycoprotein gp41, HPV-specific antigens, human telomerase reverse transcriptase, IGF I receptor, IGF-II, IL-11Rα, IL-13R-a2, influenza virus rus-specific antigens; CD38, insulin growth factor-1 (IGF1), intestinal carboxylesterase, kappa chain, LAGA-1a, lambda chain, Lassa virus-specific antigen, lectin-reactive AFP, lineage-specific antigens or tissue-specific antigens, e.g., CD3, MAGE, MAGE-A1, major histocompatibility complex (MHC) molecules, major histocompatibility complex (MHC) molecules presenting tumor-specific peptide epitopes, M-CSF, melanoma-associated antigen, mesothelin, MN-CA IX, MUC-1, mutant hsp70-2, mutant p53, mutant ras, neutrophil elastase, NKG2D, Nkp30, NY-ESO-1, p53, PAP, prostase, prostate specific antigen (PSA), prostate cancer tumor antigen-1 (PCTA-1), prostate specific antigen protein, STEAP1, STEAP2, PSMA, RAGE-1, ROR1, RU1, RU2 (AS), surface adhesion molecules, survivin and telomerase, TAG-72, fibronectin extra domain A (EDA) and extra domain B (EDB) and tenascin C A1 domain (TnC A1), thyroglobulin, tumor stromal antigen, vascular endothelial growth factor receptor-2 (VEGFR2), virus specific surface antigens, such as HIV specific antigens (e.g., HIV gp120), as well as any derivative or variant of these surface antigens.

The present disclosure is further illustrated by the following examples, which should not be construed as further limiting. The disclosure provided by this application can be used in a variety of ways, in addition to or in combination with the above methods. Below is a summary of exemplary ways that can be derived from the disclosure provided in this application.

Genetically Engineered Immune Cells and Uses In one embodiment, the cells of the present disclosure can be obtained via T cells obtained from a subject. T cells can be obtained, for example, from peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, tumors, or can be differentiated in vitro. Furthermore, T cells can be derived from one or more T cell lines available in the art. T cells can also be obtained from a unit of blood collected from a subject using various techniques known to those skilled in the art, such as FICOLL™ separation and/or apheresis. In some embodiments, cells collected by apheresis are washed to remove the plasma fraction and placed in an appropriate buffer or medium for subsequent processing. In some embodiments, the cells are washed with PBS. As will be appreciated, a washing step can be used, for example, by using a semi-automated flow-through centrifuge, such as a Cobe™ 2991 cell processing machine, Baxter CytoMate™, etc. In some embodiments, the washed cells are resuspended in one or more biocompatible buffers or other saline solutions with or without buffers. In some embodiments, undesirable components of the apheresis sample are removed. Further methods of isolating T cells for T cell therapy are disclosed in U.S. Patent Application Publication No. 2013/0287748, which is incorporated herein by reference in its entirety.

In some embodiments, T cells are isolated from PBMCs by lysing red blood cells and depleting monocytes, for example using centrifugation through a PERCOLL™ gradient. In some embodiments, specific subpopulations of T cells, such as CD4+, CD8+, CD28+, CD45RA+, and CD45RO+ T cells, are further isolated by positive or negative selection techniques known in the art. For example, enrichment of T cell populations by negative selection can be achieved using a combination of antibodies against surface markers unique to the negatively selected cells. In some embodiments, cell sorting and/or selection may be performed by negative magnetic immunoadhesion or flow cytometry using a cocktail of monoclonal antibodies against cell surface markers present on the negatively selected cells. For example, to enrich for CD4+ cells by negative selection, the monoclonal antibody cocktail typically includes antibodies against CD8, CD11b, CD14, CD16, CD20, and HLA-DR. In some embodiments, flow cytometry and cell sorting are performed to isolate cell populations of interest for use in the present disclosure.

In some embodiments, the PBMCs are used directly for genetic modification of immune cells (such as CAR) using the methods described herein. In some embodiments, after isolating the PBMCs, the T lymphocytes are further isolated and both cytotoxic and helper T lymphocytes are sorted into subpopulations of naive, memory, and effector T cells before or after genetic modification and/or expansion.

In some embodiments, CD8+ cells are further sorted into naive, central memory, and effector cells by identifying cell surface antigens associated with each of these types of CD8+ cells. In some embodiments, expression of phenotypic markers of central memory T cells includes expression of CCR7, CD3, CD28, CD45RO, CD62L, and CD127, and negativity for granzyme B. In some embodiments, central memory T cells are CD8+, CD45RO+, and CD62L+ T cells. In some embodiments, effector T cells are negative for CCR7, CD28, CD62L, and CD127, and positive for granzyme B and perforin. In some embodiments, CD4+ T cells are further classified into subpopulations. For example, CD4+ T helper cells can be sorted into naive, central memory, and effector cells by identifying cell populations with cell surface antigens.

In some embodiments, immune cells, e.g., T cells, are genetically modified after isolation using known methods, or the immune cells are activated and expanded (or differentiated in the case of progenitor cells) in vitro before being genetically modified. In another embodiment, immune cells, e.g., T cells, are genetically modified with a chimeric antigen receptor described herein (e.g., transduced with a viral vector comprising one or more nucleotide sequences encoding a CAR) and then activated and/or expanded in vitro. Methods for activating and expanding T cells are known in the art and are described, for example, in U.S. Pat. Nos. 6,905,874, 6,867,041, and 6,797,514, and WO 2012/079000, the contents of which are incorporated herein by reference in their entireties. In general, such methods involve contacting PBMCs or isolated T cells with stimulatory and costimulatory agents, such as anti-CD3 and anti-CD28 antibodies, typically bound to beads or other surfaces, in culture medium containing appropriate cytokines, such as IL-2. Anti-CD3 and anti-CD28 antibodies bound to the same bead function as "surrogate" antigen-presenting cells (APCs). One example is the Dynabeads® system, a CD3/CD28 activator/stimulator system for physiological activation of human T cells. In other embodiments, T cells are activated and stimulated to proliferate using feeder cells and appropriate antibodies and cytokines, using methods such as those described in U.S. Pat. Nos. 6,040,177 and 5,827,642 and WO 2012/129514, the contents of which are incorporated herein by reference in their entireties. In some embodiments, T cells are obtained from a donor subject. In some embodiments, the donor subject is a human patient suffering from cancer or a tumor. In some embodiments, the donor subject is a human patient not suffering from cancer or a tumor.

In one embodiment, the disclosure provides a method for producing an immunotherapy product with improved clinical efficacy and/or reduced toxicity. In some embodiments, the immunotherapy product comprises blood cells. In some embodiments, blood cells collected from a subject are washed, for example to remove the plasma fraction and place the cells in an appropriate buffer or medium for subsequent processing steps. In some embodiments, the cells are washed with phosphate buffered saline (PBS). In some embodiments, the washing solution lacks calcium and/or magnesium and/or many or all divalent cations. In some embodiments, the washing step is accomplished using a semi-automated "flow-through" centrifuge (e.g., Cobe 2991 cell processing device, Baxter) according to the manufacturer's instructions. In some embodiments, the washing step is accomplished by tangential flow filtration (TFF) according to the manufacturer's instructions. In some embodiments, the cells are resuspended in various biocompatible buffers, such as, for example, PBS without Ca++Mg++, after washing. In certain embodiments, the components of the blood cell sample are removed and the cells are resuspended directly in culture medium.

In some embodiments, the method includes a density-based cell separation method, such as preparation of leukocytes from peripheral blood by lysis of red blood cells and centrifugation through a Percoll or Ficoll density gradient. In some embodiments, the method includes leukapheresis.

In some embodiments, at least a portion of the selection step includes incubation of the cells with a selection reagent. For example, as part of the selection method, incubation with the selection reagent or reagents can be performed using one or more selection reagents for the selection of one or more different cell types based on the expression or presence of one or more specific molecules, e.g., surface markers, e.g., surface proteins, intracellular markers, or nucleic acids, in or on the cells. In some embodiments, any known method using a selection reagent or reagents for separation based on such markers can be used. In some embodiments, the selection reagent or reagents result in a separation that is an affinity or immunoaffinity based separation. For example, the selection in some embodiments includes incubation with a reagent for separation of cells and cell populations based on the expression or expression level by the cells of one or more markers, typically cell surface markers, e.g., incubation with an antibody or binding partner that specifically binds such markers, typically followed by a washing step and separation of cells that are bound to the antibody or binding partner from cells that are not bound to the antibody or binding partner.

In some embodiments of such a process, a volume of cells is mixed with a quantity of a desired affinity-based selection reagent. Immunoaffinity-based selection can be performed using any system or method that results in favorable energetic interactions between the cells to be separated and a molecule that specifically binds to a marker on the cells, e.g., an antibody or other binding partner on a solid surface, e.g., a particle. In some embodiments, the method is performed using particles such as beads, e.g., magnetic beads, that are coated with a selection agent (e.g., an antibody) specific for a marker on the cells. The particles (e.g., beads) can be incubated or mixed with the cells at a constant cell density to particle (e.g., beads) ratio that helps promote energetically favorable interactions while shaking or mixing in a container such as a tube or bag. In other cases, the method includes selection of cells where the selection is performed in whole or in part in the interior cavity of a chamber, e.g., under centrifugal rotation. In some embodiments, incubation of the cells with a selection reagent, such as an immunoaffinity-based selection reagent, is performed in the chamber.

In some embodiments, performing such a selection step or part thereof (e.g., incubation with antibody-coated particles, e.g., magnetic beads) in a cavity of a chamber allows the user to control certain parameters, e.g., the volume of various solutions, the addition of solutions during the process and their timing, which may provide advantages compared to other available methods. For example, the liquid volume in the cavity during incubation can be reduced, thereby increasing the concentration of the particles (e.g., bead reagents) used for selection, which can increase the chemical potential of the solution without affecting the total number of cells in the cavity. This can result in enhanced pairwise interactions between the cells being processed and the particles used for selection. In some embodiments, performing the incubation step in a chamber allows the user to achieve agitation of the solution at desired points during incubation, which can also improve interactions, for example when the chamber is associated with the systems, circuits, and controls described herein.

In some embodiments, at least a portion of the selection step, including incubation of the cells with the selection reagent, is carried out in a chamber. In some embodiments of such processes, a volume of cells is mixed with a much smaller amount of a selection reagent based on a desired affinity than would normally be used if a similar selection were carried out in a tube or container according to the manufacturer's instructions for the selection of the same number of cells and/or volume of cells. In some embodiments, an amount of selection reagent or selection reagents is used that is 5% or less, 10% or less, 15% or less, 20% or less, 25% or less, 50% or less, 60% or less, 70% or less, or 80% or less of the amount of the same selection reagent that would be used for the selection of cells according to the manufacturer's instructions in a tube or container-based incubation of the same number of cells and/or volume of cells.

In some embodiments, for cell selection, e.g., immunoaffinity-based selection, the cells are incubated in a composition in the chamber that also contains a selection buffer along with a selection reagent, such as an antibody optionally bound to a molecule, e.g., a scaffold, e.g., a polymer or a surface, e.g., a bead, e.g., a magnetic bead, e.g., a monoclonal antibody bound to CD4 and CD8 specific, that specifically binds to a surface marker on the cells desired to be enriched and/or removed, but not to a surface marker on other cells in the composition. In some embodiments, as described, the selection reagent is added to the cells in the cavity of the chamber in a substantially lesser amount (e.g., 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, or 80% or less) than the amount of selection reagent normally used or required to achieve about the same or comparable selection efficiency of the same number of cells or the same volume of cells when the selection is performed in a tube with shaking or rotation. In some embodiments, incubation is performed with addition of selection buffer to the cells and selection reagent to achieve a target volume for incubation of, for example, 10 mL to 200 mL, for example, at or about 10 mL, 20 mL, 30 mL, 40 mL, 50 mL, 60 mL, 70 mL, 80 mL, 90 mL, 100 mL, 150 mL, or 200 mL of reagent. In some embodiments, the selection buffer and selection reagent are premixed prior to addition to the cells. In some embodiments, the selection buffer and selection reagent are added separately to the cells. In some embodiments, the selection incubation is performed under periodic gentle mixing conditions, which may help promote energetically favorable interactions, thereby enabling the use of less selection reagent overall while achieving high selection efficiency. In some embodiments, the total duration of incubation with the selection reagent is about 5 minutes to 6 hours, e.g., 30 minutes to 3 hours, e.g., at least about 30 minutes, 60 minutes, 120 minutes, or 180 minutes, or at least about 30 minutes, 60 minutes, 120 minutes, or 180 minutes.

In some embodiments, incubation is typically performed under mixing conditions, e.g., at generally relatively low force or low speed, e.g., a speed lower than that used to pellet cells, e.g., at or about 600 rpm to 1700 rpm (e.g., 600 rpm, 1000 rpm, or 1500 rpm, or 1700 rpm, or about 600 rpm, 1000 rpm, or 1500 rpm, or 1700 rpm). , or at least 600 rpm, 1000 rpm, or 1500 rpm, or 1700 rpm), e.g., in the presence of centrifugation at 80 g to 100 g or about 80 g to 100 g (e.g., 80 g, 85 g, 90 g, 95 g, or 100 g, or about 80 g, 85 g, 90 g, 95 g, or 100 g, or at least 80 g, 85 g, 90 g, 95 g, or 100 g) of sample or RCF at the inner wall of the chamber or other container. In some embodiments, centrifugation is performed using such a low speed centrifugation period followed by a rest period, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 seconds of centrifugation and/or rest, e.g., about 1 or 2 seconds of centrifugation followed by about 5, 6, 7, or 8 seconds of rest.

In some embodiments, such a process is carried out in a completely sealed system integral to the chamber. In some embodiments, this process (and in some embodiments, one or more additional steps, e.g., a washing step immediately prior to washing a cell-containing sample, such as an apheresis sample) is carried out in an automated manner, such that cells, reagents, and other components are pumped into and pumped out of the chamber and centrifugation is accomplished at the appropriate times to complete the washing and binding steps in a single sealed system using an automated program.

In some embodiments, after incubation and/or mixing of the cells and the selection reagent and/or selection reagents, the incubated cells are subjected to separation to select cells based on the presence or absence of a particular reagent or reagents. In some embodiments, the separation is performed in the same closed system in which the incubation of the cells with the selection reagent was performed. In some embodiments, after incubation with the selection reagent, the incubated cells, including the cells to which the selection reagent is bound, are transferred to a system for immunoaffinity-based separation of the cells. In some embodiments, the system for immunoaffinity-based separation is or includes a magnetic separation column.

In some embodiments, the isolation method involves the separation of different cell types based on the expression or presence in the cells of one or more specific molecules, e.g., surface markers, e.g., surface proteins, intracellular markers, or nucleic acids. In some embodiments, any known method for such marker-based separation may be used. In some embodiments, the separation is an affinity or immunoaffinity based separation. For example, the separation in some embodiments involves the separation of cells and cell populations based on the expression or expression level of one or more markers, typically cell surface markers, by incubation with an antibody or binding partner that specifically binds to such marker, typically followed by a washing step and separation of cells that bound to the antibody or binding partner from cells that did not bind to the antibody or binding partner. Such a separation step may be based on positive selection, where cells that bound to the reagent are reserved for further use, and/or negative selection, where cells that did not bind to the antibody or binding partner are reserved. In some examples, both fractions are reserved for further use.

In some embodiments, negative selection may be particularly useful where antibodies are not available that specifically identify cell types within a heterogeneous population, such that separation is best performed based on markers expressed by cells other than the population of interest.

Separation need not result in 100% enrichment or removal of a particular cell population or cells expressing a particular marker. For example, positive selection or enrichment of a particular type of cell, e.g., cells expressing a marker, refers to increasing the number or percentage of such cells, but need not result in the complete absence of cells that do not express the marker. Similarly, negative selection, removal, or depletion of a particular type of cell, e.g., cells expressing a marker, refers to decreasing the number or percentage of such cells, but need not result in the complete removal of all such cells.

In some examples, multiple rounds of separation steps are performed in which fractions positively or negatively selected from one step are subjected to another separation step, e.g., subsequent positive or negative selection. In some examples, cells expressing multiple markers simultaneously can be removed in a single separation step, e.g., by incubating the cells with multiple antibodies or binding partners, each specific for a marker targeted by negative selection. Similarly, multiple cell types can be positively selected simultaneously by incubating the cells with multiple antibodies or binding partners expressed in different cell types.

For example, in some embodiments, specific subpopulations of T cells, e.g., cells that are positive for or express high levels of one or more surface markers, e.g., CD28+, CD62L+, CCR7+, CD27+, CD127+, CD4+, CD8+, CD45RA+, and/or CD45RO+ T cells, are isolated by positive or negative selection techniques. For example, CD3+, CD28+ T cells can be positively selected using anti-CD3/anti-CD28 conjugated magnetic beads (e.g., DYNABEADS® M-450 CD3/CD28 T Cell Expander). In some embodiments, the cell population is enriched for T cells with a naive phenotype (CD45RA+CCR7+).

In some embodiments, isolation is performed by enrichment of a particular cell population by positive selection, or depletion of a particular cell population by negative selection. In some embodiments, positive or negative selection is achieved by incubating the cells with one or more antibodies or other binding agents that specifically bind to one or more surface markers that are expressed or expressed at relatively higher levels (marker high) (marker+) on the cells being positively or negatively selected, respectively.

In certain embodiments, a biological sample, e.g., a sample of PBMCs or other white blood cells, is subjected to selection of CD4+ T cells that are retained from both the negative and positive fractions. In certain embodiments, CD8+ T cells are retained from the negative fraction. In some embodiments, a biological sample is subjected to selection of CD8+ T cells that are retained from both the negative and positive fractions. In certain embodiments, CD4+ T cells are retained from the negative fraction.

In some embodiments, T cells are separated from the PBMC sample by negative selection for a marker expressed on non-T cells, e.g., B cells, monocytes, or other leukocytes, e.g., CD14. In some embodiments, a CD4+ or CD8+ selection step is used to separate CD4+ helper and CD8+ cytotoxic T cells. Such CD4+ and CD8+ populations can be further sorted into subpopulations by positive or negative selection for markers expressed or relatively more highly expressed on one or more naive T cell, memory T cell, and/or effector T cell subpopulations.

In some embodiments, the CD8+ cells are further enriched for or further depleted of naive cells, central memory cells, effector memory cells, and/or central memory stem cells, for example, by positive or negative selection based on surface antigens associated with each subpopulation. In some embodiments, enrichment of central memory T (TcM) cells is performed to increase efficacy, e.g., to improve long-term survival, proliferation, and/or engraftment after administration, and in some embodiments efficacy is particularly strong in such subpopulations. In some embodiments, efficacy is further enhanced by combining CD8+ T cells and CD4+ T cells enriched for TcM. In some embodiments, efficacy is enhanced by enrichment of T cells with a naive phenotype (CD45RA+CCR7+). In embodiments, memory T cells are present in both the CD62L+ and CD62L subsets of CD8+ peripheral blood lymphocytes. PBMCs can be enriched for or depleted of CD62L CD8+ and/or CD62L+CD8+ fractions, for example, using anti-CD8 and anti-CD62L antibodies.

In some embodiments, the enrichment of central memory T (TCM) cells is based on positive or high surface expression of CD45RO, CD62L, CCR7, CD28, CD3, and/or CD127, and in some embodiments, this is based on negative selection of cells expressing or highly expressing CD45RA and/or granzyme B. In some embodiments, the isolation of a CD8+ population enriched in TCM cells is carried out by depletion of cells expressing CD4, CD14, CD45RA, and positive selection or enrichment of cells expressing CD62L. In one embodiment, the enrichment of central memory T (TCM) cells is carried out starting from a negative fraction of cells selected on the basis of CD4 expression, which is subjected to negative selection on the basis of expression of CD14 and CD45RA, and positive selection on the basis of CD62L. In some embodiments, such selections are carried out simultaneously, and in other embodiments, sequentially in any order. In some embodiments, the same selection step based on CD4 expression used in preparing the CD8+ cell population or subpopulation is also used to generate the CD4+ cell population or subpopulation, so that both the positive and negative fractions from the CD4-based separation are retained and used in subsequent steps of the method, optionally after one or more additional positive or negative selection steps. In a particular example, a sample of PBMCs or other leukocyte samples is subjected to selection of CD4+ cells, where both the negative and positive fractions are retained. The negative fraction is then subjected to negative selection based on expression of CD14 and CD45RA or CD19, and positive selection based on markers characteristic of central memory T cells, such as CD62L or CCR7, where the positive and negative selections are performed in either order.

CD4+ T helper cells are sorted into naive, central memory, and effector cells by identifying cell populations with cell surface antigens. CD4+ lymphocytes can be obtained by standard methods. In some embodiments, naive CD4+ T lymphocytes are CD45RO, CD45RA+, CD62L+, CD4+ T cells. In some embodiments, central memory CD4+ cells are CD62L+ and CD45RO+. In some embodiments, effector CD4+ cells are CD62L and CD45RO. In some embodiments, T cells with a naive phenotype are CD45RA+CCR7+.

In one example, a monoclonal antibody cocktail for enriching CD4+ cells by negative selection typically includes antibodies against CD14, CD20, CD11b, CD16, HLA-DR, and CD8. In some embodiments, the antibodies or binding partners are bound to a solid support or solid matrix, such as magnetic or paramagnetic beads, to allow for the separation of cells by positive and/or negative selection. For example, in some embodiments, cells and cell populations are separated or isolated using immunomagnetic (or affinity magnetic) separation techniques. In some embodiments, a sample or composition of cells to be separated is incubated with a small magnetizable or magnetically responsive material, such as a magnetically responsive particle or microparticle, such as a paramagnetic bead (e.g., Dynabeads or MACS beads). The magnetically responsive material, e.g., a particle, is typically bound directly or indirectly to a binding partner, e.g., a molecule, e.g., an antibody that specifically binds to a surface marker present on the cell, cells, or cell population that is desired to be separated, e.g., negatively or positively selected.

In some embodiments, the magnetic particles or beads include a magnetically responsive material bound to a specific binding member, such as an antibody or other binding partner. There are many known magnetically responsive materials that are used in magnetic separation methods. The incubation is typically performed under conditions in which a molecule, such as an antibody or binding partner bound to the magnetic particles or beads, or a secondary antibody or other reagent that specifically binds to such an antibody or binding partner, specifically binds to a cell surface molecule (if present on a cell in the sample). In some embodiments, the sample is placed in a magnetic field, and cells with bound magnetically responsive or magnetizable particles are attracted to the magnet and separated from unlabeled cells. Positive selection secures cells that are attracted to the magnet, and negative selection secures cells that are not attracted (unlabeled cells). In some embodiments, a combination of positive and negative selection is performed during the same selection step, and the positive and negative fractions are retained and further processed or subjected to additional separation steps. In some embodiments, the magnetically responsive particles are coated with a primary antibody or other binding partner, a secondary antibody, a lectin, an enzyme, or streptavidin. In certain embodiments, the magnetic particles are bound to the cells by coating with a primary antibody specific for one or more markers. In certain embodiments, the cells, rather than the beads, are labeled with a primary antibody or binding partner, and then magnetic particles coated with a secondary antibody or other binding partner (e.g., streptavidin) specific for the cell type are added. In certain embodiments, streptavidin-coated magnetic particles are used with biotinylated primary or secondary antibodies. In some embodiments, the magnetically responsive particles are left attached to the cells, which are then to be incubated, cultured, and/or manipulated, and in some embodiments, the particles are left attached to the cells for administration to the patient. In some embodiments, the magnetizable particles or magnetically responsive particles are removed from the cells. Methods for removing magnetizable particles from cells are known, and include, for example, the use of competitive unlabeled antibodies, and magnetizable particles or antibodies conjugated with cleavable linkers. In some embodiments, the magnetizable particles are biodegradable.

In some embodiments, affinity-based selection is by magnetically activated cell sorter (MACS) (Miltenyi Biotech, Auburn, Calif.). Magnetically activated cell sorter (MACS) systems allow for high purity selection of cells bound to magnetized particles. In certain embodiments, the MACS operates in a manner in which non-target and target species are sequentially eluted after application of an external magnetic field. That is, cells bound to magnetized particles are held in place and unbound species are eluted. Then, after this first elution step is complete, species that were trapped in the magnetic field and prevented from eluting are released in some manner such that they can be eluted and recovered. In certain embodiments, non-target cells are labeled and depleted from the heterogeneous cell population.

In some embodiments, the isolation or separation is performed using a system, device, or apparatus that performs one or more of the isolation, cell preparation, separation, processing, incubation, culture, and/or formulation steps of the method. In some embodiments, the system is used to perform each of these steps in a closed or sterile environment, e.g., to minimize errors, user handling, and/or contamination. In one example, the system is a system such as those described in WO 2009/072003 or U.S. Patent Application Publication No. 20110003380 A1. In some embodiments, the system or apparatus performs one or more, e.g., all, of the isolation, processing, manipulation, and formulation steps in an integrated or self-contained system and/or in an automated or programmable manner. In some embodiments, the system or apparatus includes a computer and/or computer program in communication with the system or apparatus that allows a user to program, control, evaluate the results of, and/or adjust various embodiments of the processing, isolation, manipulation, and formulation steps. In some embodiments, the separation and/or other steps are performed using a CliniMACS system (Miltenyi Biotec) for automated separation of cells at clinical scale levels in a closed, sterile system. Components may include an embedded microcomputer, a magnetic separation unit, a peristaltic pump, and various pinch valves. In some embodiments, the embedded microcomputer controls all components of the instrument and directs the system to perform repetitive procedures in a standardized sequence. In some embodiments, the magnetic separation unit includes a movable permanent magnet and a holder for the selected column. The peristaltic pump controls the flow rate in the tubing set and, together with the pinch valves, ensures a controlled flow of buffer through the system and continuous suspension of the cells.

In some embodiments, the CliniMACS system uses magnetizable particles coupled to antibodies, which are supplied in a sterile, non-pyrogenic solution. In some embodiments, after labeling of the cells with magnetic particles, the cells are washed to remove excess particles. The cell preparation bag is then connected to a tubing set, which in turn is connected to a bag containing buffer and a cell collection bag. The tubing set consists of pre-assembled sterile tubing, including a pre-column and a separation column, and is intended for single use only. After initiation of the separation program, the system automatically applies the cell sample onto the separation column. Labeled cells are retained in the column, while unlabeled cells are removed by a series of washing steps. In some embodiments, the cell populations used in the methods described herein are unlabeled and are not retained in the column. In some embodiments, the cell populations used in the methods described herein are labeled and are retained in the column. In some embodiments, the cell populations used in the methods described herein are eluted from the column after removal of the magnetic field and are collected in a cell collection bag.

In some embodiments, the separation and/or other steps are performed using a CliniMACS Prodigy system (Miltenyi Biotec). In some embodiments, the CliniMACS Prodigy system is equipped with a cell processing unit that allows for automated washing and fractionation of cells by centrifugation. The CliniMACS Prodigy system may also include an on-board camera and image recognition software that determines optimal cell fractionation endpoints by identifying macroscopic layers of the source cell product. For example, peripheral blood is automatically separated into red blood cell, white blood cell, and plasma layers. The CliniMACS Prodigy system may also include an integrated cell culture chamber to perform cell culture protocols such as, for example, cell differentiation and proliferation, antigen loading, and long-term cell culture. An input port may allow for the sterile removal and replenishment of media, and cells may be monitored using an integrated microscope.

In some embodiments, the cell populations described herein are collected and enriched (or depleted) by flow cytometry, in which cells stained for multiple cell surface markers are carried in a fluid stream. In some embodiments, the cell populations described herein are collected and enriched (or depleted) by preparative-scale (FACS) sorting. In certain embodiments, the cell populations described herein are collected and enriched (or depleted) by the use of a microelectromechanical system (MEMS) chip in combination with a FACS-based detection system (see, e.g., WO 2010/033140; Cho et al. (2010) Lab Chip 10, 1567-1573; and Godin et al. (2008) J Biophoton.l(5):355-376). In either case, cells can be labeled with multiple markers, allowing for the isolation of well-defined T cell subsets with high purity.

In some embodiments, the antibodies or binding partners are labeled with one or more detectable markers to facilitate separation in positive and/or negative selection. For example, separation can be based on binding to fluorescently labeled antibodies. In some examples, separation of cells based on binding of antibodies or other binding partners specific for one or more cell surface markers is performed in a fluid stream, for example by fluorescence activated cell sorting (FACS), including at a preparative scale (FACS), and/or by a microelectromechanical system (MEMS) chip in combination with, for example, a flow cytometry detection system. Such methods allow for simultaneous positive and negative selection based on multiple markers.

In some embodiments, the preparation method includes a step for freezing, e.g., cryopreserving, the cells either before or after isolation, incubation, and/or manipulation. In some embodiments, the freezing and subsequent thawing step removes granulocytes and some monocytes in the cell population. In some embodiments, the cells are suspended in a freezing solution, e.g., after a washing step to remove plasma and platelets. In some embodiments, any of a variety of known freezing solutions and parameters may be used. One example includes using PBS or other suitable cell freezing media containing 20% DMSO and 8% human serum albumin (HSA). This is then diluted 1:1 in media such that the final concentrations of DMSO and HSA are 10% and 4%, respectively. The cells are then frozen to -80°C at 1° min and stored in the vapor phase of a liquid nitrogen storage tank.

In some embodiments, the isolation and/or selection results in one or more input compositions of enriched T cells, e.g., CD3+ T cells, CD4+ T cells, and/or CD8+ T cells. In some embodiments, two or more separate input compositions are isolated, selected, enriched, or obtained from a single biological sample. In some embodiments, the separate input compositions are isolated, selected, enriched, and/or obtained from separate biological samples collected, harvested, and/or obtained from the same subject.

In certain embodiments, one or more input compositions are or comprise an enriched T cell composition that comprises at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or at or about 100% CD3+ T cells. In one embodiment, the enriched T cell input composition consists essentially of CD3+ T cells.

In certain embodiments, one or more input compositions are or comprise enriched CD4+ T cell compositions comprising at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or 100% or about 100% CD4+ T cells. In certain embodiments, the input composition of CD4+ T cells comprises less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, less than 1%, less than 0.1%, or less than 0.01% CD8+ T cells, and/or does not contain CD8+ T cells, and/or is free or substantially free of CD8+ T cells. In some embodiments, the enriched T cell composition consists essentially of CD4+ T cells.

In certain embodiments, one or more compositions are or comprise compositions of CD8+ T cells that are or comprise at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or 100% or about 100% CD8+ T cells. In certain embodiments, the composition of CD8+ T cells contains less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, less than 1%, less than 0.1%, or less than 0.01% CD4+ T cells, and/or no CD4+ T cells, and/or is free or substantially free of CD4+ T cells. In some embodiments, the enriched composition of T cells consists essentially of CD8+ T cells.

In some embodiments, the cells are incubated and/or cultured prior to or concomitantly with the genetic manipulation. The incubation step may include culturing, cultivating, stimulating, activating, and/or expanding. The incubation and/or manipulation may be performed in a culture vessel, such as a unit, chamber, well, column, tube, tube set, valve, vial, culture dish, bag, or other vessel for culturing cultures or cells. In some embodiments, the composition or cells are incubated in the presence of stimulatory conditions or stimulants. Such conditions include conditions designed to induce proliferation, expansion, activation, and/or survival of cells in the population, conditions designed to mimic antigen exposure, and/or conditions designed to prime the cells for genetic manipulation, such as the introduction of a recombinant antigen receptor. The conditions may include one or more of a particular medium, temperature, oxygen content, carbon dioxide content, time, agents, such as nutrients, amino acids, antibiotics, ions, and/or stimuli, such as cytokines, chemokines, antigens, binding partners, fusion proteins, recombinant soluble receptors, and any other agents designed to activate the cells.

In some embodiments, the stimulatory conditions or agents include one or more agents, e.g., a ligand capable of stimulating or activating an intracellular signaling domain of the TCR complex. In some embodiments, the agent activates or initiates a TCR/CD3 intracellular signaling cascade in the T cell. Such agents may include an antibody specific for the TCR, e.g., an antibody such as anti-CD3. In some embodiments, the stimulatory conditions include one or more agents, e.g., a ligand capable of stimulating a costimulatory receptor, e.g., anti-CD28. In some embodiments, such agents and/or ligands may be bound to a solid support, such as beads, and/or one or more cytokines. Optionally, the expansion method may further include adding anti-CD3 and/or anti-CD28 antibodies to the culture medium (e.g., at a concentration of at least about 0.5 ng/mL). In some embodiments, the stimulatory agents include IL-2, IL-15, and/or IL-7. In some embodiments, the IL-2 concentration is at least about 10 units/mL. In some embodiments, incubation is performed according to techniques such as those described in U.S. Pat. No. 6,040,177 to Riddell et al., Klebanoff et al. (2012) J Immunother. 35(9):651-660, Terakura et al. (2012) Blood. 1:72-82, and/or Wang et al. (2012) J Immunother. 35(9):689-701.

In some embodiments, the T cells are expanded by adding feeder cells, such as non-dividing peripheral blood mononuclear cells (PBMCs), to the culture starter composition (e.g., such that the resulting cell population contains at least about 5, 10, 20, or 40 or more PBMC feeder cells for each T lymphocyte in the initial population being expanded) and incubating the culture (e.g., for a time sufficient to expand the number of T cells). In some embodiments, the non-dividing feeder cells can include gamma-irradiated PBMC feeder cells. In some embodiments, the PBMCs are irradiated with gamma radiation in the range of about 3000-3600 rad to prevent cell division. In some embodiments, the feeder cells are added to the culture medium prior to addition of the T cell population.

In some embodiments, the stimulatory conditions include a temperature suitable for proliferation of human T lymphocytes, e.g., at least about 25 degrees Celsius, typically at least about 30 degrees Celsius, typically at or about 37 degrees Celsius. Optionally, the incubation may further include adding non-dividing EBV-transformed lymphoblastoid cells (LCL) as feeder cells. The LCL may be irradiated with gamma radiation in the range of about 6000-10,000 rad. In some embodiments, the LCL feeder cells are provided in any suitable amount, e.g., at a ratio of at least about 10:1 LCL feeder cells to initial T lymphocytes.

In embodiments, antigen-specific T cells, e.g., antigen-specific CD4+ and/or CD8+ T cells, are obtained by stimulating naive or antigen-specific T lymphocytes with an antigen. For example, antigen-specific T cell lines or clones against a cytomegalovirus antigen can be generated by isolating T cells from an infected subject and stimulating the cells in vitro with the same antigen.

In some embodiments, at least a portion of the incubation in the presence of one or more stimulating conditions or stimulants is performed in the inner cavity of a centrifuge chamber, e.g., under centrifugal rotation, e.g., as described in WO 2016/073602. In some embodiments, at least a portion of the incubation performed in the centrifuge chamber includes mixing with a reagent or reagents for inducing stimulation and/or activation. In some embodiments, cells, such as selected cells, are mixed under stimulating conditions or with stimulant agents in the centrifuge chamber. In some embodiments of such processes, a volume of cells is mixed with one or more stimulant conditions or stimulants in a much smaller amount than would normally be used when performing similar stimulation in a cell culture plate or other system.

In some embodiments, the stimulant is added to the cells in the cavity of the chamber in a substantially lesser amount (e.g., 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, or 80% or less) than the amount of stimulant normally used or required to achieve about the same or comparable selection efficiency for the same number of cells or the same volume of cells when selection is performed without mixing and with periodic shaking or rotation within the chamber, e.g., a tube or bag. In some embodiments, incubation is performed with the addition of incubation buffer to the cells and stimulants to achieve a target volume for incubation of, for example, 10 mL to 200 mL, for example, at least or at least about 10 mL, 20 mL, 30 mL, 40 mL, 50 mL, 60 mL, 70 mL, 80 mL, 90 mL, 100 mL, 150 mL, or 200 mL of reagent. In some embodiments, the incubation buffer and stimulants are premixed prior to addition to the cells. In some embodiments, the incubation buffer and stimulants are added separately to the cells. In some embodiments, the incubation for stimulation is performed under periodic gentle mixing conditions, which may help promote energetically favorable interactions, thereby enabling the use of less stimulants overall while achieving stimulation and activation of the cells.

In some embodiments, incubation is typically performed under mixing conditions, e.g., at generally relatively low forces or low speeds, e.g., speeds lower than those used to pellet cells, e.g., at or about 600 rpm to 1700 rpm (e.g., 600 rpm, 1000 rpm, or 1500 rpm, or 1700 rpm, or about 600 rpm, 1000 rpm, or 1500 rpm, or 1700 rpm). , or at least 600 rpm, 1000 rpm, or 1500 rpm, or 1700 rpm), e.g., in the presence of centrifugation at 80 g to 100 g or about 80 g to 100 g (e.g., 80 g, 85 g, 90 g, 95 g, or 100 g, or about 80 g, 85 g, 90 g, 95 g, or 100 g, or at least 80 g, 85 g, 90 g, 95 g, or 100 g) of sample or RCF at the inner wall of the chamber or other container. In some embodiments, centrifugation is performed using such a low speed centrifugation period followed by a rest period, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 seconds of centrifugation and/or rest, e.g., about 1 or 2 seconds of centrifugation followed by about 5, 6, 7, or 8 seconds of rest.

In some embodiments, for example, the total duration of incubation with the stimulant is 1 hour to 96 hours, 1 hour to 72 hours, 1 hour to 48 hours, 4 hours to 36 hours, 8 hours to 30 hours, or 12 hours to 24 hours, or about 1 hour to about 96 hours, about 1 hour to about 72 hours, about 1 hour to about 48 hours, about 4 hours to about 36 hours, about 8 hours to about 30 hours, or about 12 hours to about 24 hours, e.g., at least 6 hours, 12 hours, 18 hours, 24 hours, 36 hours, or 72 hours, or at least about 6 hours, 12 hours, 18 hours, 24 hours, 36 hours, or 72 hours. In some embodiments, the further incubation is for 1 hour to 48 hours, 4 hours to 36 hours, 8 hours to 30 hours, or 12 hours to 24 hours, or for about 1 hour to about 48 hours, about 4 hours to about 36 hours, about 8 hours to about 30 hours, or about 12 hours to about 24 hours, inclusive.

In some embodiments, the stimulatory conditions include incubating, culturing, and/or cultivating the enriched T cell composition with and/or in the presence of one or more cytokines. In certain embodiments, the one or more cytokines are recombinant cytokines. In some embodiments, the one or more cytokines are human recombinant cytokines. In certain embodiments, the one or more cytokines are expressed by the T cells and/or can bind to and/or bind to receptors endogenous to the T cells. In certain embodiments, the one or more cytokines are or include members of the four alpha helix bundle family of cytokines. In some embodiments, members of the four alpha helix bundle family of cytokines include, but are not limited to, interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin-7 (IL-7), interleukin-9 (IL-9), interleukin-12 (IL-12), interleukin-15 (IL-15), granulocyte colony stimulating factor (G-CSF), and granulocyte macrophage colony stimulating factor (GM-CSF). In some embodiments, the stimulation results in activation and/or proliferation of the cells, e.g., prior to transduction.

In some embodiments, the engineered cells, e.g., T cells, used in connection with the provided methods, uses, articles of manufacture, or compositions are cells engineered to express a recombinant receptor, e.g., a CAR or TCR as described herein. In some embodiments, the cells are engineered by introduction, delivery, or transfer of a nucleic acid sequence encoding the recombinant receptor and/or other molecule. In some embodiments, the method for making the engineered cells includes the introduction of a polynucleotide encoding a recombinant receptor (e.g., an anti-CD19 CAR) into a cell, e.g., a stimulated or activated cell. In certain embodiments, the recombinant protein is a recombinant receptor, e.g., any of those described. Introduction of a nucleic acid molecule encoding a recombinant protein, e.g., a recombinant receptor, into a cell can be carried out using any of a number of known vectors. Such vectors include viral and non-viral systems, including lentiviral and gamma retroviral systems, as well as transposon-based systems, e.g., PiggyBac or Sleeping Beauty-based gene transfer systems. Exemplary methods include those for introduction of nucleic acids encoding receptors, including by viral transduction, e.g., retroviral or lentiviral transduction, transposons, and electroporation. In some embodiments, the genetic engineering results in one or more genetically engineered compositions of enriched T cells.

In certain embodiments, one or more compositions of stimulated T cells are or include two separate stimulated compositions of enriched T cells. In some embodiments, the two separate compositions of enriched T cells, e.g., two separate compositions of enriched T cells selected, isolated, and/or enriched from the same biological sample, are genetically engineered separately. In certain embodiments, the two separate compositions include a composition of enriched CD4+ T cells. In some embodiments, the two separate compositions include a composition of enriched CD8+ T cells. In some embodiments, the two separate compositions of enriched CD4+ T cells and enriched CD8+ T cells are genetically engineered separately. In some embodiments, the same composition is enriched for CD4+ T cells and CD8+ T cells, which are genetically engineered simultaneously.

In some embodiments, the composition comprising the genetically engineered T cells comprises a pharma- ceutically acceptable carrier, diluent, solubilizer, emulsifier, preservative, and/or adjuvant. In some embodiments, the composition comprises an excipient. A "pharma-ceutically acceptable carrier" refers to an ingredient, other than an active ingredient, in a pharmaceutical formulation that is non-toxic to a subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

In some embodiments, the composition is selected for delivery via the digestive tract, such as parenterally, by inhalation, or orally. Preparation of such pharma-ceutically acceptable compositions is within the capabilities of one of ordinary skill in the art. In some embodiments, a buffer is used to maintain the composition at physiological pH or slightly lower, typically within a pH range of about 5 to about 8. In some embodiments, when parenteral administration is intended, the composition is in the form of a pyrogen-free, parenterally acceptable aqueous solution comprising the compositions described herein, with or without additional therapeutic agents, in a pharma-ceutically acceptable vehicle. In some embodiments, the parenteral injection vehicle is sterile distilled water in which the compositions described herein, with or without at least one additional therapeutic agent, are formulated as a sterile, isotonic solution and appropriately stored. In some embodiments, the preparation involves formulation of the molecule of interest with a polymeric compound (e.g., polylactic acid or polyglycolic acid), beads, or liposomes that provide a controlled or sustained release of the product, which is then delivered by depot injection. In some embodiments, the molecule of interest may be introduced using an implantable drug delivery device.

In some embodiments, the method of treating cancer in a subject in need thereof includes T cell therapy. In some embodiments, the T cell therapy disclosed herein is a genetically engineered autologous cell therapy (eACT™). According to this embodiment, the method may include harvesting blood cells from the patient. The isolated blood cells (e.g., T cells) may then be genetically engineered to express a CAR disclosed herein. In certain embodiments, the CAR T cells are administered to the patient. In some embodiments, the CAR T cells treat the tumor or cancer in the patient. In some embodiments, the CAR T cells reduce the size of the tumor or cancer.

In some embodiments, donor T cells for use in T cell therapy are obtained from a patient (e.g., for autologous T cell therapy). In other embodiments, donor T cells for use in T cell therapy are obtained from a subject that is not a patient. In certain embodiments, the T cells are tumor infiltrating lymphocytes (TILs), genetically engineered autologous T cells (eACT™), allogeneic T cells, xenogeneic T cells, or any combination thereof.

In some embodiments, the engineered T cells are administered in a therapeutically effective amount. For example, a therapeutically effective amount of engineered T cells can be at least about 10 cells, at least about ¹⁰ cells, at least about ¹⁰ cells, at least about ¹⁰ cells, at least about ¹⁰ cells, at least ^{about 10} cells, or at least about ¹⁰ cells. In ^another embodiment, a therapeutically effective amount of T cells is about ¹⁰ cells, about 10 cells, about ¹⁰ cells, about ¹⁰ cells, or about ¹⁰ cells. In some embodiments, the therapeutically effective amount of T cells is about 2×10 ⁶ cells/kg, about 3×10 ⁶ cells/kg, about 4×10 ⁶ cells/kg, about 5×10 ⁶ cells/kg, about 6×10 ⁶ cells/kg, about 7×10 ⁶ cells/kg, about 8×10 ⁶ cells/kg, about 9×10 ⁶ cells/kg, about 1×10 ⁷ cells/kg, about 2×10 ⁷ cells/kg, about 3×10 ⁷ cells/kg, about 4×10 ⁷ cells/kg, about 5×10 ⁷ cells/kg, about 6×10 ⁷ cells/kg, about 7×10 ⁷ cells/kg, about 8×10 ⁷ cells/kg, or about 9×10 ⁷ cells/kg.

In some embodiments, the therapeutically effective amount of engineered viable T cells is between about 1× ¹⁰ and about 2× ¹⁰ viable engineered T cells per kg body weight, up to a maximum dose of about 1×10 viable engineered T cells per ^kg body weight. In some embodiments, the engineered T cells are anti-CD19 CART T cells. In some embodiments, the anti-CD19 CAR T cells are an axicabtagene siloleucel product. In some embodiments, the anti-CD19 CAR T cells are a brexcabtagene autoleucel product, also known as KTE-X19.

The methods disclosed herein may be used to treat cancer in a subject, to reduce the size of a tumor, to kill tumor cells, to prevent tumor cell proliferation, to prevent tumor growth, to remove a tumor from a patient, to prevent tumor recurrence, to prevent tumor metastasis, to induce remission in a patient, or any combination thereof. In some embodiments, the methods induce a complete response. In other embodiments, the methods induce a partial response.

Cancers that may be treated include tumors that are not vascularized, tumors that are not yet substantially vascularized, or tumors that are vascularized. Cancers may also include solid or non-solid tumors. In some embodiments, the cancer is a hematological cancer. In some embodiments, the cancer is a cancer of the white blood cells. In other embodiments, the cancer is a cancer of the plasma cells. In some embodiments, the cancer is a leukemia, lymphoma, or myeloma. In some embodiments, the cancer is a cancer of the acute lymphoblastic leukemia (ALL) (including non-T cell ALL), acute lymphocytic leukemia (ALL), and hemophagocytic lymphohistiocytosis (HLH), B cell prolymphocytic leukemia, B cell acute lymphocytic leukemia ("BALL"), blastic plasmacytoid dendritic cell neoplasm, Burkitt's lymphoma, chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), chronic myelogenous leukemia (CML), chronic or acute granulomatous disease (CLL ... or acute granulomatous disease (CLL), chronic , chronic or acute leukemia, diffuse large B-cell lymphoma, diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, follicular lymphoma (FL), hairy cell leukemia, erythrophagocytic syndrome (macrophage activation syndrome (MAS)), Hodgkin's disease, large cell granuloma, leukocyte adhesion deficiency, lymphoproliferative malignancies, MALT lymphoma, mantle cell lymphoma (MCL), marginal zone lymphoma (MZL), monoclonal hypergammaglobulinemia of undetermined significance bone marrow disorders including, but not limited to, myelodysplastic syndrome (MGUS), multiple myeloma, myelodysplastic syndrome and myelodysplastic syndrome (MDS), acute myeloid leukemia (AML), non-Hodgkin's lymphoma (NHL), plasma cell proliferative disorders (e.g., asymptomatic myeloma (smoldering multiple myeloma or asymptomatic myeloma)), plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, plasmacytomas (e.g., dysplasmocytosis, solitary myeloma, solitary plasmacytoma, extramedullary plasmacytoma, and and multiple myeloma), POEMS syndrome (Crow-Fukase syndrome, Takatsuki's disease, PEP syndrome), primary mediastinal large B-cell lymphoma (PMBC), small cell or large cell follicular lymphoma, splenic marginal zone lymphoma (SMZL), systemic amyloid light chain amyloidosis, T-cell acute lymphoblastic leukemia ("TALL"), T-cell lymphoma, transformed follicular lymphoma, Waldenstrom's macroglobulinemia, or a combination thereof.

In some embodiments, the cancer is myeloma. In some embodiments, the cancer is multiple myeloma. In some embodiments, the cancer is leukemia. In some embodiments, the cancer is acute myeloid leukemia.

In some embodiments, the cancer is non-Hodgkin's lymphoma. In some embodiments, the cancer is relapsed/refractory NHL. In some embodiments, the cancer is mantle cell lymphoma.

In some embodiments, the cancer is advanced indolent non-Hodgkin's lymphoma (iNHL), including follicular lymphoma (FL) and marginal zone lymphoma (MZL). In some embodiments, the patient has developed relapsed/refractory disease after two or more prior lines of therapy, including a combination of an anti-CD20 monoclonal antibody and an alkylating agent. In some embodiments, the patient may have received a PI3K inhibitor. In some embodiments, the patient may have also received an autologous stem cell transplant. In some embodiments, patients may undergo leukapheresis to obtain T cells for CAR T cell manufacturing, followed by conditioning chemotherapy with 500 mg/m ² /day cyclophosphamide and 30 mg/m ² /day fludarabine administered on days -5, -4, and -3, and on day 0, patients may receive a single intravenous infusion of CAR T cell therapy (e.g., axicabtagene silol eucel) at a target dose of 2 x 10 ⁶ CAR T cells/kg. In some embodiments, additional infusions may be provided at subsequent periods. In some embodiments, if patients show progression after showing response at the 3 month evaluation after the first dose, patients may be re-treated with CAR T cell therapy (e.g., axicabtagene silol eucel). In some embodiments, patients may receive bridging therapy. Examples of bridging therapies include dexamethasone, rituximab, etoposide, carboplatin, ifosfamide, bendamustine-rituximab, bendamustine, methylprednisolone, mitoxantrone, cyclophosphamide, fludarabine, ibrutinib. In some embodiments, the patient experiences CRS. In some embodiments, the CRS is managed using any of the protocols described in this application, including the Examples. In some embodiments, the CRS is managed with tocilizumab, corticosteroids, and/or vasopressors.

In some embodiments, the cancer is relapsed/refractory indolent non-Hodgkin's lymphoma and the method of treating a subject in need thereof comprises administering a therapeutically effective amount of CAR T cells to the subject as a re-treatment, wherein the subject has previously received an initial treatment with CAR T cells. In some embodiments, the initial treatment with CAR T cells may have been administered as a first line or second line therapy, optionally, wherein the lymphoma is R/R follicular lymphoma (FL) or marginal zone lymphoma (MZL), and optionally, the prior prior treatment included an anti-CD20 monoclonal antibody in combination with an alkylating agent. In some embodiments, the conditioning regimen comprises an IV infusion of fludarabine 30 mg/ ^m2 and an IV infusion of cyclophosphamide 500 mg/ ^m2 on days -5, -4, and -3. In some embodiments, the CAR T cell therapy comprises a single IV infusion of 2 x 10 CAR T cells/kg on day 0. In some embodiments, at least about 10 ⁴ cells, at least about ^{10 5 cells} , at least about 10 ⁶ cells, at least about 10 ⁷ cells, at least about 10 ⁸ cells, at least about 10 ⁹ cells, or at least about 10 ¹⁰ CAR T cells are administered. In another embodiment, the therapeutically effective amount of T cells is about 10 ⁴ cells, about 10 ⁵ cells, about 10 ⁶ cells, about 10 ⁷ cells, or about 10 ⁸ cells. In some embodiments, the therapeutically effective amount of T cells is about ^2x106 cells/kg, about ^3x106 cells/kg, about ^4x106 cells/kg, about ^5x106 cells/kg, about ^6x106 cells/kg, about ^7x106 cells/kg, about ^8x106 cells/kg, about ^9x106 cells/kg, about ^1x107 cells/kg, about ^2x107 cells/kg, about ^3x107 cells/kg, about ^4x107 cells/kg, about ^5x107 cells/kg, about ^6x107 cells/kg, about ^7x107 cells/kg, about ^8x107 cells/kg, or about ^9x107 cells/kg. In some embodiments, the CAR T cells are anti-CD19 CAR T cells. In some embodiments, the CAR T cells are axicabtagene siloleucel CAR T cells. In some embodiments, the CAR T cells are brexcabtagene autoleucel/KTE-X19. In some embodiments, retreatment eligibility criteria include a CR or PR response at 3 month disease assessment followed by progression; no evidence of CD19 loss in a post-progression biopsy with local review; and/or no grade 4 CRS or neurological events or life-threatening toxicity from initial treatment with CAR T cells. In some embodiments, the method of treatment is followed by a CLINICAL TRIAL-2 clinical trial (NCT02601313). In some embodiments, the method of treatment is followed by a CLINICAL TRIAL-5 clinical trial (NCT03105336). In some embodiments, the method of treatment is followed by a CLINICAL TRIAL-6 clinical trial (NCT03105336). In some embodiments, the method of treatment is the method of treatment described in Neelapu, SS et al. 2017, N Engl J Med 2017;377(26):2531-44) and the CLINICAL TRIAL-1 (NCT02348216) clinical trial, which is described in detail in Locke, et al. Lancet Oncol. 2019;20:31-42.

In some embodiments, the cancer is NHL and the CAR T cell therapy is administered as a first line treatment. In some embodiments, the cancer is LBCL. In some embodiments, the LBCL is high-risk/high-grade LBCL with MYC and BCL2 and/or BCL6 translocations or DLBCL with an IPI score of 3 or greater at any time prior to enrollment. In some embodiments, the first line treatment comprises CAR T cell therapy in combination with a regimen comprising an anti-CD20 monoclonal antibody and an anthracycline. In some embodiments, the CAR T cell therapy is administered first. In some embodiments, the regimen comprising an anti-CD20 monoclonal antibody/anthracycline is administered first. In some embodiments, the treatments are administered at least 2 weeks, at least 4 weeks, at least 6 weeks, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, or less than 1 year apart. In some embodiments, the method further comprises bridging therapy administered after leukapheresis and completed prior to initiating conditioning chemotherapy. In some embodiments, additional inclusion criteria include age 18 years or older and ECOG PS of 0-1. In some embodiments, the conditioning therapy comprises an IV infusion of fludarabine 30 mg/ ^m2 and an IV infusion of cyclophosphamide 500 mg/ ^m2 on days -5, -4, and -3. Other exemplary beneficial preconditioning treatment regimens are described in U.S. Provisional Patent Applications Nos. 62/262,143 and 62/167,750, and U.S. Patent Nos. 9,855,298 and 10,322,146, which are incorporated herein by reference in their entireties. These describe, for example, a method of conditioning a patient in need of T cell therapy, comprising administering to said patient a specified beneficial dose of cyclophosphamide (200 mg/m ² /day to 2000 mg/m ² /day) and a specified dose of fludarabine (20 mg/m ² /day to 900 mg/m ² /day). Such a dosing regimen includes treating a patient comprising administering to the patient about 500 mg/m ² /day of cyclophosphamide and about 60 mg/m ² /day of fludarabine daily for three days prior to administering to the patient a therapeutically effective amount of engineered T cells. Another embodiment includes serum cyclophosphamide and fludarabine on days -4, -3, and -2 prior to administration of T cells, at a dose of 500 mg/m ² of cyclophosphamide per day and 30 mg/m ² of fludarabine per day during that period. Another embodiment includes cyclophosphamide on day -2 and fludarabine on days -4, -3, and -2 prior to administration of the T cells, with the cyclophosphamide at a dose of 900 mg/ ^m2 of body surface area and the fludarabine at a dose of 25 mg/ ^m2 of body surface area per day during that period. In another embodiment, conditioning includes cyclophosphamide and fludarabine on days -5, -4, and -3 prior to administration of the T cells, with the cyclophosphamide at a dose of 500 mg/ ^m2 of body surface area per day and the fludarabine at a dose of 30 mg/ ^m2 of body surface area per day during that period. Other preconditioning embodiments include cyclophosphamide at a dose of 200-300 mg/ ^m2 of body surface area per day and fludarabine at a dose of 20-50 mg/ ^m2 of body surface area per day for three days. The CLINICAL TRIAL-1 (NCT02348216) clinical trial is described in detail in Examples and in Neelapu, SS et al. 2017, N Engl J Med 2017;377(26):2531-44) and Locke, et al. Lancet Oncol. 2019;20:31-42.

In some embodiments, the treatment method involves administration of immune cells in combination with other therapeutic agents or treatments (e.g., radiation, debulking). In some embodiments, additional therapeutic agents or treatments are included to manage adverse events. In some aspects, additional therapeutic agents or treatments are included to improve the therapeutic efficacy of the cell therapy. In some instances, they accomplish both. Examples of therapeutic agents that may be used in combination with (before, after, and/or simultaneously with) the immune cells are provided below and elsewhere herein.

In some embodiments, the method further comprises administering a chemotherapeutic agent. In some embodiments, the chemotherapeutic agent selected is a lymphodepleting (preconditioning) chemotherapeutic agent. Beneficial preconditioning treatment regimens, along with correlating beneficial biomarkers, are described in U.S. Provisional Patent Applications Nos. 62/262,143 and 62/167,750, and U.S. Patent Nos. 9,855,298 and 10,322,146, which are incorporated herein by reference in their entireties. These describe, for example, methods of conditioning a patient in need of T cell therapy, comprising administering to the patient a specified beneficial dose of cyclophosphamide (200 mg/m ² /day to 2000 mg/m ² /day) and a specified dose of fludarabine (20 mg/m ² /day to 900 mg/m ² /day). Such dosing regimens include treating a patient comprising administering to the patient about 500 mg/ ^m2 /day of cyclophosphamide and about 60 mg/ ^m2 /day of fludarabine daily for three days prior to administering to the patient a therapeutically effective amount of engineered T cells. Another conditioning embodiment includes 500 mg/ ^m2 /day of cyclophosphamide and 30 mg/ ^m2 /day of fludarabine for three days on days -5, -4, and -3. Another embodiment includes serum cyclophosphamide and fludarabine on days -4, -3, and -2 prior to administration of the T cells, at a dose of 500 mg/ ^m2 of cyclophosphamide per day and 30 mg/ ^m2 of fludarabine per day during that period. Another embodiment includes cyclophosphamide on day -2 prior to administration of the T cells, and fludarabine on days -4, -3, and -2, with the cyclophosphamide dose at 900 mg/ ^m2 of body surface area and the fludarabine dose at 25 mg/ ^m2 of body surface area per day during that period. In another embodiment, conditioning includes cyclophosphamide and fludarabine on days -5, -4, and -3 prior to administration of the T cells, with the cyclophosphamide dose at 500 mg/ ^m2 of body surface area per day and the fludarabine dose at 30 mg/ ^m2 of body surface area per day during that period. Another embodiment includes cyclophosphamide at 300 mg/ ^m2 once daily for 3 days and fludarabine at 30 mg/ ^m2 once daily for 3 days. Another embodiment includes fludarabine (30 mg/ ^m2 ) for 3 days with cyclophosphamide (300 mg/ ^m2 ) given 2-7 days prior to T cell administration. Another embodiment includes cyclophosphamide at 3000 mg/m2 once daily on days -7 to -2. Another embodiment includes cyclophosphamide at 300 mg/ ^m2 once daily for ³ days and fludarabine at 30 mg/ ^m2 once daily for 3 days, with the final day occurring on days -7 to -2.

In some embodiments, the antigen binding molecule, the transduced (or otherwise engineered) cell (e.g., CAR), and the chemotherapeutic agent are each administered in an amount effective to treat the disease or condition of the subject.

In some embodiments, compositions comprising immune cells (e.g., CAR-expressing immune effector cells) disclosed herein may be administered in combination with (before, after, and/or simultaneously with administration of the immune cells) any number of other chemotherapeutic agents and/or radiation. Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosphamide (CYTOXAN™); alkylsulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodopa, carboquone, metholedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolmelamine; nitrogen mustards such as chlorambucil, chlornaphazine, chlorophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, nobembitine, phenesterine, prednimustine, trophosphamide, uracil mustard, and the like. nitrosoureas, such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine; antibiotics, such as aclacinomycin, actinomycin, ausramycin, azaserine, bleomycin, cactinomycin, calicheamicin, carabicin, carminomycin, carzinophilin, chromomycin, dactinomycin, daunorubicin, detrebicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin, idarubicin, marcellomycin, mitomycin, mycophenolic acid, nogalamycin, olivomycin, peplomycin, pofilomycin, puromycin, queramycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; antimetabolites, folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogues such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogues such as ancitabine, azacitidine, 6-azauridine, carmoful, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, 5-FU; androgens such as calsterone, dromostanolone propionate, epithiostanol, mepitiostane, testolactone; antiadrenergics such as aminoglutethimide, mitotane, trilostane; folic acid supplements such as floric acid; aceglatone; aldophosphamide glycosides; aminolevulinic acid; amsacrine, bestravcil; bisantre ; Edatrexate; Defofamine; Demecolcine; Diaziquone; Elformitin; Elliptinium acetate; Etoglucide; Gallium nitrate; Hydroxyurea; Lentinan; Lonidamine; Mitoguazone; Mitoxantrone; Mopidamol; Nitracrine; Pentostatin; Fenamet; Pirarubicin; Podophyllic acid; 2-Ethylhydrazide; Procarbazine; Polysaccharide K (PSK); Razoxane ; schizophyllan; spirogermanium; tenuazonic acid; triazicon; 2,2',2''-trichlorotriethylamine; urethane; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxoids such as paclitaxel (TAXOL™, Bristol-Myers Squibb) and doxetaxel (TAXOTERE®, Rhone-Poulenc) Rorer); chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin; xeloda; ibandronate; CPT-11; the topoisomerase inhibitor RFS2000; difluoromethylomitin (DMFO); retinoic acid derivatives such as Targretin™ (bexarotene), Panretin™ (alitretinoin); ONTAK™ (denileukin diftitox); esperamycin; capecitabine; and pharmaceutically acceptable salts, acids, or derivatives of any of the above. In some embodiments, compositions comprising CAR-expressing immune effector cells disclosed herein may be administered in conjunction with anti-hormonal agents that act to regulate or inhibit hormone action on tumors, such as anti-estrogens, including tamoxifen, raloxifene, 4(5)-imidazoles that inhibit aromatase, 4-hydroxytamoxifen, trioxyfene, keoxifene, LY117018, onapristone, and toremifene (Fareston); and anti-androgens, such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and pharmaceutically acceptable salts, acids, or derivatives of any of the above. Optionally, a combination of chemotherapy drugs is also administered, including, but not limited to, CHOP, i.e., cyclophosphamide (Cytoxan®), doxorubicin (hydroxydoxorubicin), vincristine (Oncovin®), and prednisone, R-CHOP (CHOP plus rituximab), and G-CHOP (CHOP plus obinutuzumab).

In some embodiments, the chemotherapeutic agent is administered simultaneously or within 1 week after administration of the engineered immune cells. In other embodiments, the chemotherapeutic agent is administered 1-4 weeks, or 1 week-1 month, 1 week-2 months, 1 week-3 months, 1 week-6 months, 1 week-9 months, or 1 week-12 months after administration of the engineered cells or nucleic acid. In some embodiments, the chemotherapeutic agent is administered at least 1 month prior to administration of the cells or nucleic acid. In some embodiments, the method further comprises administering two or more chemotherapeutic agents.

A variety of additional therapeutic agents may be used in conjunction with the compositions described herein (before, after, and/or concomitantly with T cell administration). For example, potentially useful additional therapeutic agents include PD-1 inhibitors, such as nivolumab (OPDIVO®), pembrolizumab (KEYTRUDA®), cemiplimab (Libtayo), pidilizumab (CureTech), and atezolizumab (Roche), and PD-L1 inhibitors, such as atezolizumab, durvalumab, and avelumab.

Additional therapeutic agents suitable for use in combination with the compositions and methods disclosed herein (before, before, after, and/or concomitantly with T cell administration) include, but are not limited to, ibrutinib (IMBRUVICA®), ofatumumab (ARZERRA®), rituximab (RITUXAN®), bevacizumab (AVASTIN®), trastuzumab (HERCEPTIN®), , trastuzumab emtansine (KADCYLA®), imatinib (GLEEVEC®), cetuximab (ERBITUX®), panitumumab (VECTIBIX®), catumaxomab, ibritumomab, ofatumumab, tositumomab, brentuximab, alemtuzumab, gemtuzumab, erlotinib, gefitinib, vandetanib, afatinib, lapatinib, neratinib, axitinib, masitinib, pazopanib , sunitinib, sorafenib, toceranib, restortinib, axitinib, cediranib, lenvatinib, nintedanib, pazopanib, regorafenib, semaxanib, sorafenib, sunitinib, tivozanib, toceranib, vandetanib, entrectinib, cabozantinib, imatinib, dasatinib, nilotinib, ponatinib, radotinib, bosutinib, restortinib, ruxolitinib, pacritinib, cobimetinib, selumetinib, trametinib, bimetinib, Nimetinib, alectinib, ceritinib, crizotinib, aflibercept, adipotide, denileukin diftitox, mTOR inhibitors such as everolimus and temsirolimus, hedgehog inhibitors such as sonidegib and vismodegib, CDK inhibitors such as the CDK inhibitor (palbociclib), inhibitors of GM-CSF, CSF1, GM-CSFR, or CSF1R, as well as antithymocyte globulin, lenzilumab, and mavrilimumab.

In one embodiment, the GM-CSF inhibitor is selected from lenzilumab; namilumab (AMG203); GSK3196165/MOR103/otilimab (GSK/MorphoSys); KB002 and KB003 (KaloBios); MT203 (Micromet and Nycomed); MORAb-022/dimcirumab (Morphotek); or a biosimilar of any of these; E21R; and a small molecule. In one embodiment, the CSF1 inhibitor is selected from RG7155, PD-0360324, MCS110/lacnotuzumab, or a biosimilar version of any of these; and a small molecule. In one embodiment, the GM-CSFR inhibitor and the CSF1R inhibitor are selected from the group consisting of mavrilimumab (formerly CAM-3001; MedImmune, Inc.); cabilalizumab (Five Prime Therapeutics); emactuzumab, also known as LY3022855 (IMC-CS4) (Eli Lilly), RG7155 or RO5509554; FPA008 (Five Prime/BMS); AMG820 (Amgen); ARRY-382 (Array Biopharma); MCS110 (Novartis); PLX3397 (Plexxikon); ELB041/AFS98/TG3003 (ElsaLys Bio, Transgene), SNDX-6352 (Syndax); biosimilar versions of any of these; and small molecules.

In some embodiments, the agents are administered by injection, e.g., intravenous or subcutaneous, intraocular, periocular, subretinal, intravitreal, transseptal, subscleral, intrachoroidal, intracameral, subconjectval, subconjuntival, subtenon, retrobulbar, peribulbar, or posterior juxtascleral delivery. In some embodiments, they are administered parenterally, intrapulmonary, and intranasally, or, if localized treatment is desired, intralesional. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous.

In some embodiments, the treatment further comprises a bridging therapy, which is a treatment between conditioning and the compositions disclosed herein, or a treatment administered after leukapheresis and completed before initiating conditioning chemotherapy. In some embodiments, the bridging therapy comprises CHOP, G-CHOP, R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisolone), corticosteroids, bendamustine, platinum compounds, anthracyclines, and/or phosphoinositide 3-kinase (PI3K) inhibitors. In some embodiments, the PI3K inhibitor is selected from duvelisib, idelalisib, venetoclax, pictilisib (GDC-0941), copanlisib, PX-866, buparlisib (BKM120), piralalisib (XL-147), GNE-317, alpelisib (BYL719), INK1117, GSK2636771, AZD8186, SAR260301, and taselisib (GDC-0032). In some embodiments, the AKT inhibitor is perifosine, MK-2206. In one embodiment, the mTOR inhibitor is selected from everolimus, sirolimus, temsirolimus, ridaforolimus. In some embodiments, the dual PI3K/mTOR inhibitor is selected from BEZ235, XL765, and GDC-0980. In some embodiments, the PI3K inhibitor is selected from duvelisib, idelalisib, venetoclax, pictilisib (GDC-0941), copanlisib, PX-866, buparlisib (BKM120), pilaralisib (XL-147), GNE-317, alpelisib (BYL719), INK1117, GSK2636771, AZD8186, SAR260301, and taselisib (GDC-0032).

In some embodiments, the bridging therapy is acalabrutinib, brentuximab vedotin, copanlisib hydrochloride, nelarabine, belinostat, bendamustine hydrochloride, carmustine, bleomycin sulfate, bortezomib, zanubrutinib, carmustine, chlorambucil, copanlisib hydrochloride, denileukin diftitox, dexamethasone, doxorubicin hydrochloride, duvelisib, pralatrexate, obinutuzumab, ibritumomab tiuxetan, ibrutinib, idelalisib, recombinant interferon alpha-2b, romidepsin, lenalidomide, mechlorethamine, These include cefotaxime hydrochloride, methotrexate, mogamulizumab-kpc, plerixafor, nelarabine, obinutuzumab, denileukin diftitox, pembrolizumab, plerixafor, polatuzumab vedotin-piiq, mogamulizumab-kpc, prednisone, rituximab, hyaluronidase, romidepsin, bortezomib, venetoclax, vinblastine sulfate, vorinostat, zanubrutinib, CHOP, COPP, CVP, EPOCH, R-EPOCH, HYPER-CVAD, ICE, R-ICE, R-CHOP, R-CVP, and combinations thereof.

In some embodiments, the cellular immunotherapy is administered in conjunction with debulking therapy used to reduce tumor burden. In one embodiment, debulking therapy should be administered after leukapheresis and prior to administration of conditioning chemotherapy or cell infusion. Examples of debulking therapy are as follows:

略語：ＡＵＣ、曲線下面積
^ａその他の減量治療選択肢が使用され得、メディカルモニターと共に協議される。現地の基準に従って、水分補給、制吐剤、メスナ、増殖因子支持薬、及び腫瘍崩壊予防による支持療法が使用され得る。１クール超が許可される。
^ｂ腫瘍測定を可能にするために、少なくとも１つの標的病変を放射線照射野外に残す。

Abbreviations: AUC, area under the curve
^aOther debulking treatment options may be used and will be discussed with the medical monitor. Supportive care with hydration, antiemetics, mesna, growth factor support, and oncolytic prophylaxis may be used according to local standards. More than one course is permitted.
^bAt least one target lesion is left outside the radiation field to allow for tumor measurements.

In some embodiments, compositions comprising immune cells (e.g., engineered CAR T cells) are administered with an anti-inflammatory agent (either prior to administration of the immune cells, after administration of the immune cells, and/or simultaneously with administration of the immune cells). Anti-inflammatory agents or drugs include, but are not limited to, steroids and glucocorticoids (including betamethasone, budesonide, dexamethasone, hydrocortisone acetate, hydrocortisone, hydrocortisone, methylprednisolone, prednisolone, prednisone, triamcinolone), nonsteroidal anti-inflammatory drugs (NSAIDs), including aspirin, ibuprofen, naproxen, methotrexate, sulfasalazine, leflunomide, anti-TNF drugs, cyclophosphamide, and mycophenolates. Exemplary NSAIDs include ibuprofen, naproxen, naproxen sodium, Cox-2 inhibitors, and sialylates. Exemplary analgesics include acetaminophen, oxycodone, proporoxifene hydrochloride, tramadol.Exemplary glucocorticoids include cortisone, dexamethasone, hydrocortisone, methylprednisolone, prednisolone, or prednisone. Exemplary biological response modifiers include molecules against cell surface markers (e.g., CD4, CD5, etc.), cytokine inhibitors such as TNF antagonists (e.g., etanercept (ENBREL®), adalimumab (HUMIRA®) and infliximab (REMICADE®), chemokine inhibitors, and adhesion molecule inhibitors. Biological response modifiers include monoclonal antibodies as well as recombinant forms of molecules. Exemplary DMARDs include azathioprine, cyclophosphamide, cyclosporine, methotrexate, penicillamine, leflunomide, sulfasalazine, hydroxychloroquine, Gold (oral (auranofin) and intramuscular), and minocycline.

In some embodiments, the compositions described herein are administered with a cytokine (either prior to administration of the T cells, after administration of the T cells, or simultaneously with administration of the T cells). Examples of cytokines are lymphokines, monokines, and traditional polypeptide hormones. Cytokines include growth hormones, e.g., human growth hormone, N-methionyl human growth hormone, and bovine growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoprotein hormones, e.g., follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), luteinizing hormone (luteinizing hormone), and the like. hormone, LH); hepatic growth factor (HGF); fibroblast growth factor (FGF); prolactin; placental lactogen; murelan inhibitor; mouse gonadotropin-related peptide; inhibin; activin; vascular endothelial growth factor; integrin; thrombopoietin (TPO); nerve growth factor (NGF), e.g., NGF-beta; platelet growth factor; transforming growth factor (TGF), e.g., TGF-α and TGF-β; insulin-like growth factor-I and growth factor-II; erythropoietin (EPO, Epogen®, Procrit®); bone morphogenetic factor; interleukin-1 (IL-1); Interferons, such as interferon-alpha, beta, and gamma; colony stimulating factors (CSF), such as macrophage-CSF (M-CSF); granulocyte-macrophage CSF (GM-CSF); and granulocyte CSF (G-CSF); interleukins (IL), such as IL-1, IL-1 alpha, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12; IL-15, tumor necrosis factors, such as TNF-alpha or TNF-beta; and other polypeptide factors, including LIF and Kit Ligand (KL). As used herein, the term cytokine includes proteins from natural sources or from recombinant cell culture and biologically active equivalents of the native sequence cytokines.

In some embodiments, administration of the cells and administration of the additional therapeutic agent are performed on the same day, or are performed no more than 36 hours apart, no more than 24 hours apart, no more than 12 hours apart, no more than 6 hours apart, no more than 4 hours apart, no more than 2 hours apart, or no more than 1 hour apart, or no more than 30 minutes apart. In some embodiments, administration of the cells and administration of the additional therapeutic agent are between 0 hours or about 0 hours and 48 hours or about 48 hours, between 0 hours or about 0 hours and 36 hours or about 36 hours, between 0 hours or about 0 hours and 24 hours or about 24 hours, between 0 hours or about 0 hours and 12 hours or about 12 hours, between 0 hours or about 0 hours and 6 hours or about 6 hours, between 0 hours or about 0 hours and 2 hours or about 2 hours, between 0 hours or about 0 hours and 1 hour or about 1 hour, between 0 hours or about 0 hours and 30 minutes or about 30 minutes, between 30 minutes or about 30 minutes and 48 hours or about 48 hours, between 30 minutes or about 30 minutes and 36 hours or about 36 hours, between 30 minutes or about 30 minutes and 36 hours or about 36 hours, between 30 minutes or about 30 minutes between or about 24 hours, between or about 30 minutes and 12 hours, between or about 30 minutes and 6 hours, between or about 30 minutes and 4 hours, between or about 30 minutes and 2 hours, between or about 30 minutes and 1 hour, between or about 1 hour and 48 hours, between or about 1 hour and 36 hours, between or about 1 hour and 24 hours, between or about 1 hour and 12 hours, between or about 1 hour and 6 hours, between or about 1 hour and 4 hours, between an hour or about 1 hour and 2 hours or about 2 hours, between 2 hours or about 2 hours and 48 hours or about 48 hours, between 2 hours or about 2 hours and 36 hours or about 36 hours, between 2 hours or about 2 hours and 24 hours or about 24 hours, between 2 hours or about 2 hours and 12 hours or about 12 hours, between 2 hours or about 2 hours and 6 hours or about 6 hours, between 2 hours or about 2 hours and 4 hours or about 4 hours, between 4 hours or about 4 hours and 48 hours or about 48 hours, between 4 hours or about 4 hours and 36 hours or about 36 hours, between 4 hours or about 4 hours and 24 hours or about 24 hours, between 4 hours or about 4 hours and 12 hours or about 12 hours, between 4 hours or about 4 hours The administration may be performed for 6 hours or about 6 hours, between 6 hours or about 6 hours and 48 hours or about 48 hours, between 6 hours or about 6 hours and 36 hours or about 36 hours, between 6 hours or about 6 hours and 24 hours or about 24 hours, between 6 hours or about 6 hours and 12 hours or about 12 hours, between 12 hours or about 12 hours and 48 hours or about 48 hours, between 12 hours or about 12 hours and 36 hours or about 36 hours, between 12 hours or about 12 hours and 24 hours or about 24 hours, between 24 hours or about 24 hours and 48 hours or about 48 hours, between 24 hours or about 24 hours and 36 hours or about 36 hours, or between 36 hours or about 36 hours and 48 hours or about 48 hours. In some embodiments, the cells and the additional therapeutic agent are administered simultaneously.

In some embodiments, the agent is administered in a dosage of 30 mg to 5000 mg or about 30 mg to 5000 mg, such as 50 mg to 1000 mg, 50 mg to 500 mg, 50 mg to 200 mg, 50 mg to 100 mg, 100 mg to 1000 mg, 100 mg to 500 mg, 100 mg to 200 mg, 200 mg to 1000 mg, 200 mg to 500 mg, or 500 mg to 1000 mg. In some embodiments, the agent is administered at a dosage of 0.5 mg/kg to 100 mg/kg, 1 mg/kg to 50 mg/kg, 1 mg/kg to 25 mg/kg, 1 mg/kg to 10 mg/kg, 1 mg/kg to 5 mg/kg, 5 mg/kg to 100 mg/kg, 5 mg/kg to 50 mg/kg, 5 mg/kg to 25 mg/kg, 5 mg/kg to 10 mg/kg, 10 mg/kg to 100 mg/kg, 10 mg/kg to 50 mg/kg, 10 mg/kg to 25 mg/kg, 25 mg/kg to 100 mg/kg, 25 mg/kg to 50 mg/kg, 50 mg/kg to 100 mg/kg. In some embodiments, the agent is administered at a dose of 1 mg/kg to 10 mg/kg, 2 mg/kg to 8 mg/kg, 2 mg/kg to 6 mg/kg, 2 mg/kg to 4 mg/kg, or 6 mg/kg to 8 mg/kg, respectively. In some aspects, the agent is administered at a dose of at least 1 mg/kg, 2 mg/kg, 4 mg/kg, 6 mg/kg, 8 mg/kg, 10 mg/kg, or more.

Response and Efficacy Measurements In some embodiments, the methods described herein may provide a clinical benefit to a subject. In some embodiments, at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of patients achieve a clinical benefit. In some embodiments, about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 0%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of patients achieve a clinical benefit, and any unrecited percentages therebetween. In some embodiments, the response rate is 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 9.5%, 10.5%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 109%, 109%, 102%, 104%, 105%, 106%, 107%, 108%, 109%, 109%, 109%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, or other unrecited percentages and ranges from 1% to 100%. In some embodiments, the response rate is 0%-10%, 10%-20%, 20%-30%, 30%-40%, 40%-50%, 50%-60%, 60%-70%, 70%-80%, 80%-90%, or 90%-100%. In some embodiments, the response rate is 0%-1%, 1%-1.5%, 1.5%-2%, 2%-3%, 3%-4%, 4%-5%, 5%-6%, 6%-7%, 7%-8%, 8%-9%, 9%-10%, 10%-15%, 15%-20%, 20-25%, 25%-30%, 35-40%, or other various ranges up to 95%-100%.

The clinical benefit may be an objective response or a sustained clinical response, defined as a sustained response at a median follow-up of one year. In some embodiments, the response, blood levels of CAR T cells, or immune-related factors are determined at about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, or about 7 days after administration of the engineered CAR T cells. In some embodiments, the response, blood levels of CAR T cells, or immune-related factors are determined at about 1 week, about 2 weeks, about 3 weeks, or about 4 weeks after administration of the engineered CAR T cells. In some embodiments, the response, blood levels of CAR T cells, and/or immune-related factors are determined by follow-up at about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, about 13 months, about 14 months, about 15 months, about 16 months, about 17 months, about 18 months, about 19 months, about 20 months, about 21 months, about 22 months, about 23 months, or about 24 months after administration of the engineered CAR T cells. In some embodiments, the response, blood levels of CAR T cells, and/or immune-related factors are determined by follow-up at about 1 year, about 1.5 years, about 2 years, about 2.5 years, about 3 years, about 4 years, or about 5 years after administration of the engineered CAR T cells.

Monitoring In some embodiments, administration of the chimeric receptor T cell immunotherapy is performed at a certified medical center.

In some embodiments, the methods disclosed herein include monitoring the patient daily for at least 7 days after infusion at a qualified medical facility for signs and symptoms of CRS and neurotoxicity and other adverse reactions to the CAR T cell therapy. In some embodiments, the symptoms of neurotoxicity are selected from encephalopathy, headache, tremors, dizziness, aphasia, delirium, insomnia, and anxiety. In some embodiments, the symptoms of adverse reactions are selected from the group consisting of fever, hypotension, tachycardia, hypoxia, and chills, including cardiac arrhythmias (including atrial fibrillation and ventricular tachycardia), cardiac arrest, heart failure, renal failure, capillary leak syndrome, hypotension, hypoxia, organ toxicity, hemophagocytic lymphohistiocytosis/macrophage activation syndrome (HLH/MAS), seizures, encephalopathy, headache, tremors, dizziness, aphasia, delirium, insomnia, anxiety, anaphylaxis, febrile neutropenia, thrombocytopenia, neutropenia, and anemia. In some embodiments, patients are instructed to remain near a qualified medical facility for at least four weeks after the infusion.

Clinical Outcome In some embodiments, the clinical outcome is a complete response. In some embodiments, the clinical outcome is a durable response. In some embodiments, the clinical outcome is a complete response. In some embodiments, the clinical outcome is not a response. In some embodiments, the clinical outcome is a partial response. In some embodiments, the clinical outcome is an objective response. In some embodiments, the clinical outcome is survival. In some embodiments, the clinical outcome is recurrence.

In some embodiments, objective response (OR) is determined according to the revised IWG Response Criteria for Lymphoma (Cheson, 2007) and determined by the IWG Response Criteria for Lymphoma (Cheson et al. Journal of Clinical Oncology 32, no. 27 (September 2014) 3059-3067). Duration of response (DOR) is defined only for subjects experiencing an objective response and is the time from first objective response to disease progression (per Cheson et al, 2014) or disease-related death, whichever occurs first. Progression-free survival (PFS) is assessed by investigator assessment according to the Lugano response criteria.

Prevention or Management of Severe Adverse Reactions In some embodiments, the disclosure provides a method of preventing the onset of an adverse reaction or reducing the severity of an adverse reaction based on the level of one or more characteristics. In some embodiments, the cell therapy is administered with one or more agents that prevent, delay the onset, reduce symptoms, or treat an adverse event, including cytokine release syndrome and neurotoxicity. In one embodiment, the agents are described above. In other embodiments, the agents are described below. In some embodiments, the agents are administered before, after, or simultaneously with administration of the cells, in one of the methods and dosages described elsewhere herein. In one embodiment, the agents are administered to a subject who may be predisposed to a disease, but has not yet been diagnosed with the disease.

In this regard, the disclosed methods may include administration of a "prophylactically effective amount" of tocilizumab, corticosteroid therapy, and/or an anticonvulsant for toxicity prevention. In some embodiments, the methods include administering an inhibitor of GM-CSF, CSF1, GM-CSFR, or CSF1R, lenzilumab, mavrilimumab, a cytokine, and/or an anti-inflammatory agent. The pharmacological and/or physiological effect may be prophylactic, i.e., the effect completely or partially prevents a disease or its symptoms. A "prophylactically effective amount" may refer to an amount effective to achieve a desired prophylactic result (e.g., prevention of the onset of an adverse reaction) at the dosage and duration required.

In some embodiments, the method includes managing an adverse reaction in any subject. In some embodiments, the adverse reaction is selected from the group consisting of cytokine release syndrome (CRS), neurotoxicity, hypersensitivity reactions, serious infections, cytopenias, and hypogammaglobulinemia.

In some embodiments, the signs and symptoms of the adverse reaction are selected from the group consisting of fever, hypotension, tachycardia, hypoxia, and chills, and include cardiac arrhythmias (including atrial fibrillation and ventricular tachycardia), cardiac arrest, heart failure, renal insufficiency, capillary leak syndrome, hypotension, hypoxia, organ toxicity, hemophagocytic lymphohistiocytosis/macrophage activation syndrome (HLH/MAS), seizures, encephalopathy, headache, tremor, dizziness, aphasia, delirium, insomnia anxiety, anaphylaxis, febrile neutropenia, thrombocytopenia, neutropenia, and anemia.

In some embodiments, patients are identified and selected based on one or more of the biomarkers described in this application. In some embodiments, patients are identified and selected simply by clinical symptoms (e.g., the presence and grade of toxic symptoms).

Cytokine Release Syndrome (CRS)
In some embodiments, the methods include preventing or reducing the severity of CRS in a chimeric receptor therapy, hi some embodiments, the engineered immune cells (e.g., CAR T cells) are inactivated after administration to the patient.

In some embodiments, the method includes identifying CRS based on clinical symptoms. In some embodiments, the method includes evaluating and treating other causes of fever, hypoxia, and hypotension. Patients with Grade 2 or higher CRS (e.g., hypotension unresponsive to fluid infusion or hypoxia requiring supplemental oxygen) should be monitored with continuous electrocardiogram telemetry and pulse oximetry. In some embodiments, patients with severe CRS should be considered for performing an echocardiogram to assess cardiac function. In cases of severe or life-threatening CRS, supportive care with intensive care may be considered.

In some embodiments, the method includes monitoring the patient daily for at least 7 days after the infusion at a qualified medical facility for signs and symptoms of CRS. In some embodiments, the method includes monitoring the patient for signs or symptoms of CRS for 4 weeks after the infusion. In some embodiments, the method includes advising the patient to seek immediate medical attention whenever signs or symptoms of CRS occur. In some embodiments, the method includes initiating treatment with supportive care, tocilizumab, or tocilizumab and corticosteroids as indicated at the first sign of CRS.

Neurotoxicity (NT)
In some embodiments, the method includes monitoring the patient for signs and symptoms of neurotoxicity. In some embodiments, the method includes excluding other causes of neurological symptoms. Patients experiencing grade 2 or higher neurotoxicity should be monitored with continuous cardiac telemetry and pulse oximetry. In cases of severe or life-threatening neurotoxicity, intensive supportive care is provided. In some embodiments, symptoms of neurotoxicity are selected from encephalopathy, headache, tremors, dizziness, aphasia, delirium, insomnia, and anxiety.

Management of Adverse Events In some embodiments, the cell therapy is administered before, during/concurrently with, and/or after administration of one or more agents (e.g., steroids) or treatments (e.g., weight loss) that treat or prevent one or more symptoms of an adverse event (prophylactic). A "prophylactically effective amount" refers to an amount effective to achieve a desired prophylactic result at the dosage and duration required. In one embodiment, a prophylactically effective amount is used in a subject prior to or at an early stage of disease. In one embodiment, a prophylactically effective amount will be less than a therapeutically effective amount. In some embodiments, a patient is selected for management of an adverse event based on expression of one or more of the markers described herein. In one embodiment, treatment or prevention of an adverse event is administered to any patient who will undergo, is undergoing, or has undergone cell therapy.

In some embodiments, the method of managing adverse events includes monitoring the patient daily for at least 7 days after the infusion at a qualified medical facility for signs and symptoms of neurotoxicity. In some embodiments, the method includes monitoring the patient for signs or symptoms of neurotoxicity and/or CRS for 4 weeks after the infusion.

In some embodiments, the disclosure provides two methods of managing adverse events in subjects receiving CAR T cell therapy with steroids and anti-IL6/anti-IL-6R antibodies. In one embodiment, the methods are described in FIG. 46. In one embodiment, the disclosure provides that early steroid intervention in cohort 4 is associated with a lower rate of severe CRS and neurological events compared to that observed in cohorts 1+2. In one embodiment, the disclosure provides that early use of steroids in cohort 4 resulted in a median cumulative cortisone equivalent dose of about 15% of that in cohorts 1+2, suggesting that early steroid use may allow for a reduction in total steroid exposure. Thus, in one embodiment, the disclosure provides a method of managing adverse events in which corticosteroid therapy is initiated for management of all cases of grade 1 CRS if there is no improvement after 3 days for all grade 1 or higher neurological events. In one embodiment, the disclosure provides a method of reducing total steroid exposure in patients undergoing adverse event management following CAR T cell administration, comprising initiating corticosteroid therapy for management of all cases of grade 1 CRS if all grade ≧1 neurological events have not improved after 3 days, and/or initiating tocilizumab for all cases of grade 1 CRS if all grade ≧2 neurological events have not improved after 3 days. In one embodiment, the corticosteroid and tocilizumab are administered in a regimen selected from those illustrated in Table 12. In one embodiment, the disclosure provides that early steroid use is not associated with an increased risk of severe infection, decreased proliferation of CAR T cells, or decreased tumor response.

In one embodiment, the disclosure supports the safety of levetiracetam prophylaxis in CAR T cell cancer treatment. In one embodiment, the cancer is NHL. In one embodiment, the cancer is R/R LBCL and the patient is administered axicabtagene siloleucel. Thus, in one embodiment, the disclosure provides a method of managing adverse events in a patient treated with immune cells (e.g., CAR T cells), comprising administering a prophylactic dose of an anticonvulsant to the patient. In some embodiments, the patient is administered levetiracetam (e.g., 750 mg orally or intravenously twice daily) starting on day 0 of CAR T cell treatment (post-pretreatment) and at the onset of grade >2 neurotoxicity if a neurological event occurs after discontinuation of prophylactic levetiracetam. In one embodiment, if the patient does not experience any grade >2 neurotoxicity, levetiracetam is tapered and discontinued as clinically indicated. In one embodiment, levetiracetam prophylaxis is combined with any other adverse event management protocol.

In one embodiment, the disclosure provides that CAR T cell levels in patients subject to the Cohort 4 adverse event management protocol were comparable to those in Cohort 1+2. In one embodiment, the disclosure provides that the numerical levels of inflammatory cytokines (e.g., IFNγ, IL-2, and GM-CSF) associated with CAR-associated inflammatory events are lower in Cohort 4 than in Cohort 1+2. Thus, the disclosure provides a method of reducing CAR T cell therapy-associated inflammatory events without affecting CAR T cell levels, comprising administering to a patient an adverse event management protocol of Cohort 4. The disclosure also provides a method of reducing immune cell cytokine production following CAR T cell therapy, comprising administering to a patient an adverse event management protocol of Cohort 4. In one embodiment, this effect is achieved without affecting CAR T cell expansion and response rates. In one embodiment, the patient is afflicted with R/R LBCL. In one embodiment, the CAR T cell therapy is an anti-CD19 CAR T cell therapy. In one embodiment, the CAR T cell therapy includes axicabtageneciloleucel.

In one embodiment, the disclosure provides that early or prophylactic use of tocilizumab after axicabtagene ciloreucel for adverse event management reduced grade 3 or higher cytokine release syndrome but increased grade 3 or higher neurological events. Thus, the disclosure provides a method for adverse event management in CAR T cell therapy as described in FIG. 56. In one embodiment, the patient is administered levetiracetam (750 mg twice daily, orally or intravenously) starting on day 0. Upon onset of grade 2 or higher neurological events, the dose of levetiracetam is increased to 1000 mg twice daily. If the patient does not exhibit any grade 2 or higher neurological events, levetiracetam is tapered and discontinued as clinically necessary. The patient is also administered tocilizumab (8 mg/kg IV over 1 hour [not to exceed 800 mg]) on day 2. Further tocilizumab (± corticosteroids) may be recommended at the onset of grade 2 CRS in patients with comorbidities or elderly patients, or otherwise in case of grade 3 or higher CRS. Tocilizumab is initiated for patients with grade 2 or higher neurological events, and corticosteroids are added for patients with comorbidities or elderly patients, or in the event of a grade 3 or higher neurological event that worsens despite the use of tocilizumab.

In one embodiment, the disclosure provides that prophylactic use of steroids appears to reduce the rate of severe CRS and NE to the same extent as early steroid use after axicabtagene silolucel administration. Thus, the disclosure provides a method for adverse event management in CAR T cell therapy, in which a patient is administered 10 mg of dexamethasone orally on days 0 (before axicabtagene silolucel infusion), 1, and 2. Steroids are also administered upon the occurrence of grade 1 NE and for grade 1 CRS if no improvement is observed after 3 days of supportive care. Tocilizumab is also administered for grade 1 or higher CRS if no improvement is observed after 24 hours of supportive care.

In one embodiment, the disclosure provides that adverse event management of CAR T cell therapy with an antibody that neutralizes and/or depletes GM-CSF prevents or reduces treatment-related CRS and/or NE in treated patients. In one embodiment, the antibody is lenzilumab.

In some embodiments, the adverse events are managed by administration of an agent/agents that are antagonists or inhibitors of IL-6 or the IL-6 receptor (IL-6R). In some embodiments, the agent is an antibody that neutralizes IL-6 activity, e.g., an antibody or antigen-binding fragment that binds to IL-6 or IL-6R. For example, in some embodiments, the agent is or includes the anti-IL-6R antibodies tocilizumab (atlizumab) or sarilumab. In some embodiments, the agent is an anti-IL-6R antibody described in U.S. Pat. No. 8,562,991. In some examples, the agent targeting IL-6 is an anti-TL-6 antibody, such as siltuximab, elcilimomab, ALD518/BMS-945429, sirukumab (CNTO 136), CPSI-2634, ARGX 109, FE301, FM101, or olokizumab (CDP6038), and combinations thereof. In some embodiments, the agent may neutralize IL-6 activity by inhibiting the ligand-receptor interaction. In some embodiments, the IL-6/IL-6R antagonist or inhibitor is an IL-6 mutein, such as those described in U.S. Pat. No. 5,591,827. In some embodiments, the agent that is an antagonist or inhibitor of IL-6/IL-6R is a small molecule, a protein or peptide, or a nucleic acid.

In some embodiments, other agents that may be used to manage adverse reactions and their symptoms include cytokine receptor or cytokine antagonists or inhibitors. In some embodiments, the cytokine or receptor is IL-10, TL-6, TL-6 receptor, IFNy, IFNGR, IL-2, IL-2R/CD25, MCP-1, CCR2, CCR4, MIP13, CCR5, TNFα, TNFR1, e.g., TL-6 receptor (IL-6R), IL-2 receptor (IL-2R/CD25), MCP-1 (CCL2) receptor (CCR2 or CCR4), TGFβ receptor (TGFβI, II, or III), IFNγ receptor (IFNGR), MIP1P receptor (e.g., CCR5), TNFα receptor (e.g., TNFR1), IL-1 receptor (IL1-Ra/IL-1RP), or IL-10 receptor (IL-10R), IL-1, and IL-1Rα/IL-1β. In some embodiments, the agent comprises siltuximab, sarilumab, olokizumab (CDP6038), elcilimomab, ALD518/BMS-945429, sirukumab (CNTO 136), CPSI-2634, ARGX 109, FE301, or FM101. In some embodiments, the agent is a cytokine antagonist or inhibitor, for example, transforming growth factor beta (TGFβ), interleukin 6 (TL-6), interleukin 10 (IL-10), IL-2, MIP13 (CCL4), TNFα, IL-1, interferon gamma (IFN-γ), or monocyte chemotactic protein-1 (MCP-1). In some embodiments, the agent targets (e.g., inhibits or is an antagonist of) a cytokine receptor, such as TL-6 receptor (IL-6R), IL-2 receptor (IL-2R/CD25), MCP-1 (CCL2) receptor (CCR2 or CCR4), TGFβ receptor (TGFβI, II, or III), IFNγ receptor (IFNGR), MIP1P receptor (e.g., CCR5), TNFα receptor (e.g., TNFR1), IL-1 receptor (IL1-Ra/IL-1RP), or IL-10 receptor (IL-10R), and combinations thereof. In some embodiments, the agent is administered before, after, or simultaneously with administration of the cells, by one of the methods and doses described elsewhere herein.

In some embodiments, the agent is administered at a dose of 1 mg/kg to 10 mg/kg or about 1 mg/kg to 10 mg/kg, 2 mg/kg to 8 mg/kg or about 2 mg/kg to 8 mg/kg, 2 mg/kg to 6 mg/kg or about 2 mg/kg to 6 mg/kg, 2 mg/kg to 4 mg/kg or about 2 mg/kg to 4 mg/kg, or 6 mg/kg to 8 mg/kg or about 6 mg/kg to 8 mg/kg, inclusive, or the agent is administered at a dose of at least 2 mg/kg or at least about 2 mg/kg or about 2 mg/kg, at least 4 mg/kg or at least about 4 mg/kg or about 4 mg/kg, at least 6 mg/kg or at least about 6 mg/kg or about 6 mg/kg, or at least 8 mg/kg or at least about 8 mg/kg or about 8 mg/kg. In some embodiments, the agent is administered at a dose of about 1 mg/kg to 12 mg/kg, e.g., 10 mg/kg or about 10 mg/kg. In some embodiments, the agent is administered by intravenous infusion. In one embodiment, the agent is tocilizumab. In some embodiments, the agent, e.g., particularly tocilizumab, is administered before, after, or simultaneously with administration of the cells, by one of the methods and doses described elsewhere herein.

In some embodiments, the method includes identifying CRS based on clinical symptoms. In some embodiments, the method includes evaluating and treating other causes of fever, hypoxia, and hypotension. If CRS is observed or suspected, it may be managed according to the recommendations of Protocol A, which may be used in combination with other therapies disclosed herein, including neutralization or reduction of the CSF/CSFR1 axis. Patients exhibiting Grade 2 or higher CRS (e.g., hypotension unresponsive to fluid infusion or hypoxia requiring supplemental oxygen) should be monitored with continuous electrocardiogram telemetry and pulse oximetry. In some embodiments, patients exhibiting severe CRS should be considered for performing an echocardiogram to assess cardiac function. In cases of severe or life-threatening CRS, supportive care with intensive care may be considered. In some embodiments, a biosimilar or equivalent of tocilizumab may be used in place of tocilizumab in the methods disclosed herein. In other embodiments, another anti-IL6R may be used in place of tocilizumab.

In some embodiments, adverse events are managed according to the following protocol (Protocol A):

（ａ）Ｌｅｅ ＤＷら（２０１４）．Ｃｕｒｒｅｎｔ ｃｏｎｃｅｐｔｓ ｉｎ ｔｈｅ ｄｉａｇｎｏｓｉｓ ａｎｄ ｍａｎａｇｅｍｅｎｔ ｏｆ ｃｙｔｏｋｉｎｅ ｒｅｌｅａｓｅ ｓｙｎｄｒｏｍｅ．Ｂｌｏｏｄ．２０１４ Ｊｕｌ １０；１２４（２）：１８８－１９５。
（ｂ）神経毒性の管理については表２を参照されたい。
（ｃ）詳細については、ＡＣＥＭＴＲＡ（登録商標）（トシリズマブ）処方情報、ｈｔｔｐｓ：／／ｗｗｗ．ｇｅｎｅ．ｃｏｍ／ｄｏｗｎｌｏａｄ／ｐｄｆ／ａｃｔｅｍｒａ＿ｐｒｅｓｃｒｉｂｉｎｇ．ｐｄｆ（最終アクセス日２０１７年１０月１８日）。最初の米国の承認は２０１０年に示されている。

(a) Lee DW et al. (2014). Current concepts in the diagnosis and management of cytokine release syndrome. Blood. 2014 Jul 10;124(2):188-195.
(b) See Table 2 for management of neurotoxicity.
(c) For more information, see ACEMTRA® (tocilizumab) Prescribing Information, https://www.gene.com/download/pdf/actemra_prescribing.pdf (last accessed October 18, 2017). First U.S. Approval for this was announced in 2010.

Neurotoxicity In some embodiments, the method includes monitoring the patient for signs and symptoms of neurotoxicity. In some embodiments, the method includes excluding other causes of neurological symptoms. Patients experiencing grade 2 or higher neurotoxicity should be monitored with serial cardiac telemetry and pulse oximetry. In cases of severe or life-threatening neurotoxicity, provide intensive supportive care. Consider a non-sedating anti-seizure drug (e.g., levetiracetam) for seizure prophylaxis for any grade 2 or higher neurotoxicity. The following treatments can be used in combination with other treatments of the present disclosure, such as neutralization or reduction of the CSF/CSFR1 axis.

In some embodiments, adverse events are managed according to the following protocol (Protocol B):

Additional safety management measures with corticosteroids Administration of corticosteroids and/or tocilizumab in Grade 1 can be considered prophylactic. Supportive care can be provided in all protocols for all CRS and NE severity grades.

In one embodiment of the protocol for managing adverse events associated with CRS, tocilizumab and/or corticosteroids, the following are administered: Grade 1 CRS: no tocilizumab; no corticosteroids; Grade 2 CRS: tocilizumab (only if comorbidities or elderly); and/or corticosteroids (only if comorbidities or elderly); Grade 3 CRS: tocilizumab; and/or corticosteroids; Grade 4 CRS: tocilizumab; and/or corticosteroids. In another embodiment of the protocol for managing adverse events associated with CRS, tocilizumab and/or corticosteroids are administered as follows: Grade 1 CRS: tocilizumab (if no improvement after 3 days); and/or corticosteroids (if no improvement after 3 days); Grade 2 CRS: tocilizumab; and/or corticosteroids; Grade 3 CRS: tocilizumab; and/or corticosteroids; Grade 4 CRS: tocilizumab; and/or corticosteroids, high dose.

In one embodiment of the protocol for managing adverse events associated with NE, tocilizumab and/or corticosteroids, the following are administered: Grade 1 NE: no tocilizumab; no corticosteroids; Grade 2 NE: no tocilizumab; no corticosteroids; Grade 3 NE: tocilizumab; and/or corticosteroids (standard dose only if no improvement to tocilizumab); Grade 4 NE: tocilizumab; and/or corticosteroids.

In another embodiment of the protocol for managing adverse events associated with NE, tocilizumab and/or corticosteroids are administered as follows: Grade 1 NE: no tocilizumab; and/or corticosteroids; Grade 2 NE: tocilizumab; and/or corticosteroids; Grade 3 NE: tocilizumab; and/or corticosteroids, high dose; Grade 4 NE: tocilizumab; and/or corticosteroids, high dose.

In one embodiment, corticosteroid treatment is initiated at grade 2 or higher CRS and tocilizumab is initiated at grade 2 or higher CRS. In one embodiment, corticosteroid treatment is initiated at grade 1 or higher CRS and tocilizumab is initiated at grade 1 or higher CRS. In one embodiment, corticosteroid treatment is initiated at grade 3 or higher NE and tocilizumab is initiated at grade 3 or higher CRS. In one embodiment, corticosteroid treatment is initiated at grade 1 or higher CRS and tocilizumab is initiated at grade 2 or higher CRS. In some embodiments, prophylactic use of tocilizumab administered on day 2 may reduce the rate of grade 3 or higher CRS.

In one embodiment, adverse events may be managed by a method including Protocol C.

^ａ治験担当医師の裁量で症状の改善時に漸減される治療法；^ｂ８００ｍｇを超えないこと；ＡＥは、有害事象、ＣＲＳは、サイトカイン放出症候群、ＩＶは、静脈内、Ｎ／Ａは、該当せず、ＮＥは、神経学的事象を示す。

^a Treatment tapered upon symptomatic improvement at investigator's discretion; ^b Not to exceed 800 mg; AE, adverse event, CRS, cytokine release syndrome, IV, intravenous, N/A, not applicable, NE, neurological event.

Any corticosteroid may be suitable for this use. In one embodiment, the corticosteroid is dexamethasone. In some embodiments, the corticosteroid is methylprednisolone. In some embodiments, a combination of the two is administered. In some embodiments, glucocorticoids include synthetic and non-synthetic glucocorticoids. Exemplary glucocorticoids include alclomethasone, alginate, beclomethasone (e.g., beclomethasone dipropionate), betamethasone (e.g., betamethasone 17-valerate, betamethasone sodium acetate, betamethasone sodium phosphate, betamethasone valerate), budesonide, clobetasol (e.g., clobetasol propionate), clobetasone, clocortolone (e.g., clocortolone pivalate), cloprednol, corticosterone, cortisone and hydrocortisone (e.g., hydrocortisone acetate), cortivazol, deflazacol, desonide, desoximetasone, dexamethasone (e.g., dexamethasone 21 phosphate, dexamethasone acetate, dexamethasone sodium phosphate), Diflorasone (e.g., diflorasone diacetate), diflucortralone, difluprednate, enoxolone, furazacor, fluclonide, fludrocortisone (e.g., fludrocortisone acetate), flumethasone (e.g., flumethasone pivalate), flunisolide, fluocinolone (e.g., fluocinolone acetonide), fluocinonide, flucortin, flutrolone, fluorometholone (e.g., fluorometholone acetate), fluperolone (e.g., fluperone acetate), fluprednidene, fluprednisolone, flurandrenolide, fluticasone (e.g., fluticasone propionate), formocortal, halcinonide, halobetasol, halometasone, halopredone, hydrocortamate, hydrocortisone (e.g., hydrocortisone 21 - butyrate, hydrocortisone aceponate, hydrocortisone acetate, hydrocortisone buteprate, hydrocortisone butyrate, hydrocortisone cypionate, hydrocortisone hemisuccinate, hydrocortisone probutate, hydrocortisone sodium phosphate, hydrocortisone sodium succinate, hydrocortisone valerate), loteprednol etabonate, mazipredone, medrysone, meprednisone, methylprednisolone (methylprednisolone aceponate, methylprednisolone acetate, methylprednisolone hemisuccinate, methylprednisolone sodium succinate), mometasone (e.g. Mometasone Furoate), paramethasone (e.g. Paramethasone Acetate), prednicarbate, prednisolone (e.g. Predni prednisolone 25-diethylaminoacetate, prednisolone sodium phosphate, prednisolone 21-hemisuccinate, prednisolone acetate; prednisolone farnesylate, prednisolone hemisuccinate, prednisolone-21 (beta-D-glucuronide), prednisolone metasulfobenzoate, prednisolone stearate, prednisolone tebutate, prednisolone tetrahydrophthalate), prednisone, prednival, prednylidene, rimexolone, tixocortol, triamcinolone (e.g., triamcinolone acetonide, triamcinolone benetonide, triamcinolone hexacetonide, triamcinolone acetonide 21 palmitate, triamcinolone diacetate). These glucocorticoids and their salts are described in detail, for example, in Remington's Pharmaceutical Sciences, A. Osol, ed., Mack Pub. Co., Easton, Pa. (16th ed. 1980) and Remington: The Science and Practice of Pharmacy, 22nd Edition, Lippincott Williams & Wilkins, Philadelphia, Pa. (2013), as well as any other editions, which are incorporated herein by reference. In some embodiments, the glucocorticoid is selected from among cortisone, dexamethasone, hydrocortisone, methylprednisolone, prednisone, and prednisone. In one embodiment, the glucocorticoid is dexamethasone. In another embodiment, the steroid is a mineralocorticoid. Any other steroid may be used in the methods provided herein.

The one or more corticosteroids may be administered at any dose and dosing frequency that may be compatible with the severity/grade of the adverse event (e.g., CRS and NE). Tables 1 and 2 provide examples of dosing regimens for managing CRS and NE, respectively. In another embodiment, the administration of the corticosteroid comprises dexamethasone 10 mg administered orally or IV 1-4 times per day. Another embodiment, sometimes referred to as a "high-dose" corticosteroid, comprises methylprednisone 1 g/day administered IV, alone or in combination with dexamethasone. In some embodiments, the one or more corticosteroids are administered at a dose of 1-2 mg/kg per day.

The corticosteroid may be administered in any amount effective to ameliorate one or more symptoms associated with an adverse event, such as CRS or neurotoxicity. Corticosteroids, e.g., glucocorticoids, may be administered in an amount of at or about 0.1-100 mg, 0.1-80 mg, 0.1-60 mg, 0.1-40 mg, 0.1-30 mg, 0.1-20 mg, 0.1-15 mg, 0.1-10 mg, 0.1-5 mg, 0.2-40 mg, 0.2-30 mg, 0.2-20 mg, 0.2-15 mg, 0.2-10 mg, 0.2-5 mg, 0.4-40 mg, 0.4-30 mg, 0.4-20 mg, 0.4-15 mg, 0.4-10 mg, 0.4-5 mg, 0.4-4 mg, 1-20 mg, 1-15 mg, or 1-10 mg per dose, for example, to a 70 kg adult human subject. Typically, corticosteroids, such as glucocorticoids, are administered to an average adult human subject in an amount of about 0.4 to 20 mg per dose, e.g., about 0.4 mg, 0.5 mg, 0.6 mg, 0.7 mg, 0.75 mg, 0.8 mg, 0.9 mg, 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 11 mg, 12 mg, 13 mg, 14 mg, 15 mg, 16 mg, 17 mg, 18 mg, 19 mg, or 20 mg.

In some embodiments, the corticosteroid is administered at a dose of, for example, 0.001 or about 0.001 mg/kg (of subject), 0.002 mg/kg, 0.003 mg/kg, 0.004 mg/kg, 0.005 mg/kg, 0.006 mg/kg, 0.007 mg/kg, 0.008 mg/kg, 0.009 mg/kg, 0.01 mg/kg, 0.015 mg/kg, 0.02 mg/kg, 0.025 mg/kg, 0.03 mg/kg, 0.035 mg/kg, 0.04 mg/kg, 0.045 mg/kg, 0.05 mg/kg, 0.055 mg/kg, 0.06 mg/kg, 0.065 mg/kg, 0.07 mg/kg, 0.075 mg/kg, for an average adult subject, typically weighing about 70 kg to 75 kg. , 0.08mg/kg, 0.085mg/kg, 0.09mg/kg, 0.095mg/kg, 0.1mg/kg, 0.15mg/kg, 0.2mg/kg, 0.25mg/kg, 0.30mg/kg, 0.35mg/kg, 0.40mg/kg, 0.45mg/kg, 0.50mg/kg, 0.55mg/kg, 0.60mg/kg, 0.65mg/kg g, 0.70 mg/kg, 0.75 mg/kg, 0.80 mg/kg, 0.85 mg/kg, 0.90 mg/kg, 0.95 mg/kg, 1 mg/kg, 1.05 mg/kg, 1.1 mg/kg, 1.15 mg/kg, 1.20 mg/kg, 1.25 mg/kg, 1.3 mg/kg, 1.35 mg/kg, or 1.4 mg/kg.

In general, the dose of corticosteroid administered will depend on the particular corticosteroid, as differences in potency exist between different corticosteroids. Typically, drugs vary in potency, with the understanding that doses may differ to achieve equivalent effect. Equivalence in terms of potency for various glucocorticoids and routes of administration is well known. Information regarding equivalent steroid doses (non-chronotherapeutic) can be found in the British National Formulary (BNF) 37, March 1999.

In some embodiments, adverse events are managed by the following protocol: patients receive levetiracetam (750 mg orally or intravenously twice daily) starting on day 0 of administration of T cell therapy; upon onset of grade 2 or higher neurological events, the levetiracetam dose is increased to 1000 mg twice daily; if the patient does not experience any grade 2 or higher neurological events, levetiracetam is tapered and discontinued as clinically necessary; patients also receive tocilizumab (8 mg/kg) on day 2. IV over 1 hour [not to exceed 800 mg]); additional tocilizumab (± corticosteroids) may be recommended at the onset of grade 2 CRS in patients with comorbidities or elderly patients, or otherwise in case of grade 3 or higher CRS; tocilizumab is initiated for patients with grade 2 or higher neurological events, and corticosteroids are added for patients with comorbidities or elderly patients, or if a grade 3 or higher neurological event occurs that worsens despite the use of tocilizumab. In some embodiments, levetiracetam is administered for prophylaxis and at the onset of grade 2 or higher neurotoxicity if a neurological event occurs after discontinuation of levetiracetam prophylaxis, and/or levetiracetam is tapered and discontinued if the patient does not show any grade 2 or higher neurotoxicity.

In some embodiments, adverse events are managed by the following protocol: patients receive 10 mg of dexamethasone orally on days 0 (prior to infusion of T cell therapy), 1, and 2; steroids are also administered upon occurrence of grade 1 NE and for grade 1 CRS if no improvement is observed after 3 days of supportive care; tocilizumab is also administered for grade 1 or greater CRS if no improvement is observed after 24 hours of supportive care.

Secondary Malignancies In some embodiments, patients treated with immune cells (e.g., CAR T cells) or other genetically modified autologous T cell immunotherapies (e.g., targeting CD19) may develop secondary malignancies. In certain embodiments, patients treated with CAR T cells (e.g., targeting CD19) or other genetically modified allogeneic T cell immunotherapies may develop secondary malignancies. In some embodiments, the method includes lifelong monitoring for secondary malignancies.

Example 1
This example provides results from an analysis of the CLINICAL TRIAL-2 clinical trial. The study design of the CLINICAL TRIAL-2 trial is summarized in Figure 1. Safety and efficacy results and pharmacokinetic profiles among patients with and without disease progression within 24 months of diagnosis (POD24) are reported here. Disease progression within 24 months of initial diagnosis (POD24) is an indicator of poor outcome in patients with mantle cell lymphoma (MCL) (Visco C, et al. Br J Haematol. 2019; 185: 940-944). In a retrospective analysis of MCL patients, median overall survival (OS) from the time of progression was 12 months for POD24 patients, whereas non-POD24 patients were not reached. KTE-X19, an autologous anti-CD19 chimeric antigen receptor (CAR) T cell therapy, is approved in the United States and European Union for the treatment of relapsed/refractory (R/R) MCL (TECARTUS® (brexucabtagene autolucel) Prescribing Information. Kite Pharma, Inc; 2021; TECARTUS® (autologous anti-CD19 transduced CD3+ cells) Summary of Product Characteristics. Kite Pharma EU B.V.; 2021). The pivotal Phase 2 CLINICAL TRIAL-2 study evaluated KTE-X19 in MCL patients who were R/R to 1-5 prior therapies, including a Bruton's tyrosine kinase inhibitor (BTKi) (Wang M, et al. N Engl J Med. 2020;382:1331-1342). After a median follow-up of 17.5 months in CLINICAL TRIAL-2 (N=60), the objective response rate (ORR) was 92% and the complete response (CR) rate was 67% (Wang M, et al. Blood. 2020;136(Suppl 1):20-22). 48% of all patients and 70% of CR patients were ongoing at the data cut-off date. Cytokine release syndrome (CRS) and neurological events (NE) were mostly reversible (N=68 treated patients). 15% of patients had grade 3 or higher CRS, whereas 31% had grade 3 or higher NE. Two patients had grade 5 adverse events (AEs), only one of which was KTE-X19-related. No new safety signals were observed with longer follow-up (Wang M, et al. N Engl J Med. 2020; 382: 1331-1342, Wang M, et al. Blood. 2020; 136 (Suppl 1): 20-22).

Eligible patients were 18 years of age or older with pathologically confirmed mantle cell lymphoma (MCL) with either documented cyclin D1 overexpression or the presence of t(11;14) or relapsed/refractory to 1-5 prior regimens for MCL. Prior therapy included anthracycline or bendamustine-containing chemotherapy, anti-CD20 monoclonal antibodies, and ibrutinib or acalabrutinib. All patients received prior BTKi. Patients must have received prior BTKi therapy, but this need not have been their most recent therapy prior to study entry, and patients did not need to be refractory to BTKi therapy. Eligible patients had an absolute lymphocyte count of ≥100/μL. Patients who had received autologous SCT within 6 weeks of CD19 CAR-T infusion or had previously received CD19-targeted therapy or allogeneic SCT were excluded. All patients underwent leukapheresis to obtain cells for the manufacture of CD19 CAR-T cell therapeutics. Patients received optional bridging therapy including dexamethasone (20-40 mg or equivalent), ibrutinib (560 mg once daily orally (PO)), or acalabrutinib (100 mg twice daily PO). The manufacturing process for KTE-X19 was modified for axicabtageneciloleucel to remove circulating lymphoma cells by positive enrichment of CD4 ⁺ /CD8 ⁺ cells. This product is referred to herein as "CAR T cells." Conditioning chemotherapy with fludarabine (30 mg/m ² /day) and cyclophosphamide (500 mg/m ² /day) was administered on days -5, -4, and -3, followed by a single intravenous infusion of CD19 CAR-T cells at 2 x 10 ⁶ CAR T cells/kg on day 0.

This example reports safety outcomes, pharmacological profile, and product attributes for all 68 patients treated with KTE-X19. Efficacy results are reported in 60 treated patients with more than 1 year of follow-up (median 17.5 months). Data are presented with a data cut-off date of December 31, 2019.

Patient baseline characteristics are summarized in Table 1. High-risk disease characteristics were common in POD24 and non-POD24 patients. POD24 patients had higher tumor burden and lactate dehydrogenase (LDH) levels, and were more likely to have blastoid MCL, suggesting that these patients may be less well-matched than non-POD24 patients. POD24 patients were more likely to have high-risk disease characteristics (high tumor burden, high LDH levels, and blastoid MCL) than non-POD24 patients.

Overall response rates (ORR; CR, and partial response) were assessed by an independent radiological review committee according to the Lugano classification (Cheson BD et al., Journal of clinical oncology 2014;32:3059-68). ORR was similar between patients on POD24 and not on POD24, with a slightly higher CR rate in patients on POD24 (Figure 2). After a median follow-up of 17.5 months, KTE-X19 produced high CR rates in patients on POD24 and not on POD24. Minimal residual disease (MRD; sensitivity ^10-5 ) was assessed by next-generation sequencing as previously reported (Wang M, et al. New Engl J Med. 2020;382:1331-1342). MRD was assessed in patients with available samples at week 4. Similar rates of MRD negativity were also observed among patients on POD 24 (75%, n=9/12) and not on POD 24 (79%, n=15/19).

Secondary efficacy-related endpoints included duration of response (DOR), progression-free survival (PFS), and overall survival (OS) (Figure 3). Median progression-free survival (PFS) was 11.3 months (95% CI, 6.0-NE) in patients with POD 24. Median PFS appeared to be shorter among patients with POD 24 compared with patients without POD 24. Median duration of response (DOR) and OS were not reached in either group (Figure 3).

The safety profiles of POD24 and non-POD24 patients were generally similar. The incidence of grade ≥3 adverse events was generally similar between POD24 and non-POD24 patients (Table 2). The incidence of thrombocytopenia and neutropenia appeared to be higher in POD24 patients than in non-POD24 patients. The incidence of infections appeared to be higher in non-POD24 patients than in POD24 patients. There were no cases of grade 5 cytokine release syndrome, KTE-X19-associated secondary malignancies, or proliferative retrovirus in either group.

^ａＣＲＳは、Ｌｅｅ ＤＷ，ｅｔ ａｌ．Ｂｌｏｏｄ．２０１４；１２４：１８８－１９５に従ってグレード分けした。ＣＲＳの症状及び全ての他のＡＥは、米国国立がん研究所の有害事象共通用語規準４．０３版に従ってグレード分けした。ＡＥは、有害事象、ＣＲＳは、サイトカイン放出症候群、ＰＯＤ２４の場合、疾患の進行は初回診断後２４か月未満であり、ＰＯＤ２４ではない場合、疾患の進行は初回診断後２４か月以上である。

^a CRS was graded according to Lee DW, et al. Blood. 2014;124:188-195. CRS symptoms and all other AEs were graded according to the National Cancer Institute's Common Terminology Criteria for Adverse Events version 4.03. AE = adverse event, CRS = cytokine release syndrome, if POD24, disease progression <24 months after initial diagnosis, if not POD24, disease progression ≥24 months after initial diagnosis.

Product attributes and blood CAR T cell levels were analyzed using methods previously described (Locke FL, et al. Mol Ther. 2017;25:285-295). KTE-X19 product characteristics were similar between POD 24 and non-POD 24 patients (Table 3). POD 24 patients appeared to have lower CAR T cell expansion than non-POD 24 patients. In POD 24 patients, the median peak CAR T cell levels and median area under the curve (AUC) were 53.4 cells/μL (range, 0.2-2566) and 583.4 cells/μL x days (range, 1.8-27, 743.6; Figure 4). Patients not on POD24 had median peak CAR T cell levels and median AUC of 112.4 cells/μL (range, 0.2-2589) and 1588.3 cells/μL x days (range, 3.8-27,238.7).

Among efficacy-evaluable patients with available data, B cells were detectable by 12 months in 8/11 (73%) patients with POD 24 and 7/15 (47%) patients without POD 24 (Figure 5). Early intervention with CD19-targeted CAR T cell therapy may benefit MCL patients with known high-risk factors, such as POD 24 (Visco C, et al. Br J Haematol. 2019;185:940-944).

Example 2
This example provides results from an analysis of the CLINICAL TRIAL-5 clinical trial, a phase 2, multicenter, single-arm study of axicabtagene siloleucel in patients with relapsed/refractory (R/R) indolent non-Hodgkin lymphoma (R/R iNHL, NCT03105336). The study design of the CLINICAL TRIAL-5 trial is summarized in FIG. Axi-cel is an autologous anti-CD19 chimeric antigen receptor (CAR) T-cell therapy approved in the United States (US) for the treatment of adults with relapsed/refractory (R/R) FL after two or more lines of systemic therapy and in the US and European Union for adults with R/R large B-cell lymphoma (LBCL) after two or more lines of systemic therapy (YESSCARTA® (Axi-cel) Prescribing Information. Kite Pharma, Inc; 2021; YESSCARTA® (Axi-cel) [Summary of Product Characteristics]. Amsterdam, The Netherlands: Kite Pharma EU B.V.; 2018).

Progression within 24 months after starting the first anti-CD20-containing chemoimmunotherapy (POD24) is a risk factor for poor survival in patients with indolent non-Hodgkin lymphoma (iNHL) (Casulo C and Barr P. Blood. 2019; 133(14): 1540-154; Casulo C, et al. J Clin Oncol. 2015; 33(23): 2516-2522). Approximately 20% of patients with follicular lymphoma (FL) are POD24. In an observational analysis of the National LymphoCare Study, patients with early-progressing FL had a lower 5-year overall survival (OS) rate (50%) than those without early progression (90%). This is a report of safety and efficacy results and pharmacokinetic/pharmacodynamic profiles with longer follow-up between POD 24 and non-POD 24 patients in CLINICAL TRIAL-5.

Adults with follicular lymphoma (FL) (grade 1-3a) or marginal zone lymphoma (MZL, nodal or extranodal) had R/R disease after 2 or more lines of therapy (including anti-CD20 mAb + alkylating agent) and ECOG 0-1. Patients received leukapheresis followed by conditioning therapy (intravenous fludarabine (30 mg/ ^m2 body surface area) and cyclophosphamide (500 mg/ ^m2 body surface area) on days -5, -4, and -3) and a single infusion of axicabtagene ciloreucel at ^2x106 CAR T cells/kg on day 0. The primary endpoint was objective response rate (ORR) (complete response (CR) + partial response (PR)) as assessed by central review (according to the Lugano classification; Cheson BD, et al. J Clin Oncol. 2014;32(27):3059-3068. doi:10.1200/JCO.2013.54.8800). Secondary endpoints included complete response (CR) rate (according to the Lugano classification; Cheson, et al. J Clin Oncol. 2014), duration of response (DOR) (DOR is defined only for subjects experiencing an objective response and is the time from the first objective response to disease progression (Cheson et al., 2014) or disease-related death, whichever occurs first), progression-free survival (PFS) (PFS is defined as the time from the date of axicabtagene siloleucel infusion to disease progression (Cheson et al., 2014) or death from any cause), overall survival (OS) (OS is defined as the time from axicabtagene siloleucel infusion to the date of death), incidence of adverse events (AEs), and blood CAR T cell and serum cytokine levels. The primary efficacy analysis was performed when 80 or more FL-treated patients had a follow-up of 12 months or more. At a median follow-up of 17.5 months in the primary analysis (Jacobson et al. ASH 2020. #700), 92% of patients responded (76% complete response [CR] rate). The grade 3 or higher neurological event (NE; MZL) rate in FL patients was lower (15%) than in MZL patients (41%). In the primary analysis, the overall response rate (ORR) after a median follow-up of 17.5 months was similar between patients with POD24 and those without POD24 (93% vs. 92%).

The updated efficacy analysis was performed when 80 or more FL-treated patients had ≥18 months of follow-up. Efficacy evaluable patients included ≥80 FL-treated patients with ≥18 months of follow-up after axicabtagene siloleucel infusion and ≥4 weeks of follow-up after axicabtagene siloleucel infusion as of the data cut-off date (September 14, 2020). FL or MZL patients treated with axicabtagene siloleucel and available data on progression after anti-CD20 mAb + alkylating agent were included in the POD24 analysis (N=129).

Baseline characteristics were generally similar between POD24 and non-POD24 patients (Table 4). Among the FL patients evaluated, median tumor burden by sum of two-way products (SPD) was numerically similar in POD24 and non-POD24 patients (2303 ^mm2 vs. 2839 ^mm2 ). Among the MZL patients evaluated, median SPD appeared to be higher among POD24 patients than non-POD24 patients (2028 ^mm2 vs. 954 ^mm2 ).

^ａＧＥＬＦ基準によって定義される疾患負荷：３以上の結節部位（それぞれ直径３ｃｍ以上）を伴う；直径７ｃｍ以上の任意の結節性又は結節外腫瘍塊；Ｂ症状；巨脾腫；胸水又は腹水；血球減少症；又は白血病。^ｂ１種類の先行治療を受けた３人のＦＬ患者の登録は、２種類以上の先行治療を要するプロトコール修正の前にＣＬＩＮＩＣＡＬ ＴＲＩＡＬ－５で行われた。^ｃ直近の先行治療の完了の６か月以内に進行したｉＮＨＬ患者。ＦＬは、濾胞性リンパ腫、ＦＬＩＰＩは、濾胞性リンパ腫国際予後指標、ＧＥＬＦは、Ｇｒｏｕｐｅ ｄ’Ｅｔｕｄｅ ｄｅｓ Ｌｙｍｐｈｏｍｅｓ Ｆｏｌｌｉｃｕｌａｉｒｅｓ；ＭＺＬは、辺縁帯リンパ腫、ＰＩ３Ｋｉは、ホスホイノシチド３－キナーゼ阻害剤、ＰＯＤ２４の場合、疾患の進行は最初の抗ＣＤ２０含有化学免疫療法を開始してから２４か月未満であり、ＳＣＴは、幹細胞移植を意味する。

aDisease burden defined by GELF criteria: ≥3 nodal sites (each ≥3 cm in diameter); any nodal or extranodal mass ≥7 cm in diameter; B symptoms; splenomegaly; pleural or ascites effusion; cytopenia; or leukemia. ^bThree FL patients with one prior therapy were enrolled in CLINICAL TRIAL-5 prior to the protocol amendment requiring ≥2 prior therapies. cPatients with iNHL ^who progressed within 6 months of completion of ^most recent prior therapy. FL, follicular lymphoma; FLIPI, Follicular Lymphoma International Prognostic Index; GELF, Groupe d'Etude des Lymphomes Follicularis; MZL, marginal zone lymphoma; PI3Ki, phosphoinositide 3-kinase inhibitor; POD24, disease progression is less than 24 months after starting initial anti-CD20-containing chemoimmunotherapy; SCT, stem cell transplant.

Secondary efficacy-related endpoints included duration of response (DOR), progression-free survival (PFS), and overall survival (OS). Overall response rate (ORR; CR, and partial response) was assessed by an independent radiological review committee according to the Lugano classification (Cheson BD et al., Journal of clinical oncology 2014;32:3059-68). ORR was similar between efficacy-evaluable patients on POD 24 and those not on POD 24 (Figure 7; Table 5). Estimated median duration of response (DOR) was not reached after a median follow-up of 17.1 and 17.5 months, respectively, in patients on POD 24 and those not on POD 24 (Figure 8A, Figure 8B, Figure 8C; Table 5). 52% of efficacy-evaluable patients on POD24 and 70% of efficacy-evaluable patients on non-POD24 were in response at the data cut-off date. 18-month DOR rates in POD24 and non-POD24 patients were 60% and 78%, respectively. Median progression-free survival (PFS) and OS were not reached in POD24 and non-POD24 patients (Figures 8B, 8C; Table 5). 18-month PFS rates in POD24 and non-POD24 patients were 55% and 84%, respectively. 18-month OS rates were 85% and 94%, respectively.

ＣＲは、完全奏効、ＤＯＲ、奏功期間；ＦＬは、濾胞性リンパ腫、ｍｏは、月数、ＭＺＬは、辺縁帯リンパ腫、ＮＤは、未実施／未定義、ＮＥは、推定不可、ＮＲは、未到達、
ＯＳは、全生存期間ＰＦＳは、無増悪生存期間、ＰＯＤ２４の場合、疾患の進行は最初の抗ＣＤ２０含有化学免疫療法を開始してから２４か月未満であり、ＰＲは、部分奏効を意味する。

CR, complete response; DOR, duration of response; FL, follicular lymphoma; mo, months; MZL, marginal zone lymphoma; ND, not performed/not defined; NE, not estimable; NR, not reached;
OS is overall survival, PFS is progression-free survival, POD24 means disease progression less than 24 months after starting the first anti-CD20-containing chemoimmunotherapy, and PR means partial response.

The incidence of grade 3 or higher adverse events was generally similar between POD24 and non-POD24 patients (Table 6). Grade 5 events, including one event in the setting of cytokine release syndrome (CRS), occurred in three POD24 patients. No grade 5 events occurred in non-POD24 patients. One POD24 patient experienced grade 4 CRS. Grade 4 neurological events occurred in two POD24 patients. No non-POD24 patients experienced grade 4 CRS or neurological events.

CAR T cell levels in blood, cytokine levels in serum, and product attributes and their association with clinical outcomes were analyzed by using previously described methods (Locke FL, et al. Mol Ther. 2017;25:285-295).

In efficacy-evaluable FL patients, median peak CAR T cell levels were similar in POD24 and non-POD24 patients (36.9 cells/μL and 34.5 cells/μL, respectively; Figure I9A). Median AUC was also similar between POD24 and non-POD24 patients (422.5 cells/μL×days and 407.6 cells/μL×days, respectively (Figure 9B). Pretreatment levels of CCL17 (TARC) and CCL22 (MDC) were higher in POD24 patients than in non-POD24 patients. Peak levels of biomarkers associated with axicabtageneciloleucel toxicity were generally similar in all POD24 and non-POD24 treated patients (Table 7). Pharmacokinetic/pharmacodynamic findings between groups were similar in MZL patients.

Ｐ値は、ウィルコクソンの順位和検定を使用して計算した。^ａ使用したアッセイにおける定量化の下限。^ｂ使用したアッセイにおける定量化の上限。^ｃピークでは、２人のＦＬ患者のデータが入手不能であった。ＣＣＬは、ケモカイン（Ｃ－Ｃモチーフ）リガンド、ＣＲＰは、Ｃ反応性タンパク質、ＣＸＣＬ、Ｃ－Ｘ－Ｃモチーフケモカインリガンド；ＦＬは、濾胞性リンパ腫、ＧＭ－ＣＳＦは、顆粒球マクロファージコロニー刺激因子、ＩＦＮは、インターフェロン、ＩＬは、インターロイキン、ＭＣＰ－１は、単球走化性タンパク質１、ＰＯＤ２４の場合、疾患の進行は最初の抗ＣＤ２０含有化学免疫療法を開始してから２４か月未満であり、ＲＡは、受容体アンタゴニスト、ＳＡＡは、血清アミロイドＡ、ＴＮＦは、腫瘍壊死因子を意味する。

P values were calculated using the Wilcoxon rank sum test. ^a Lower limit of quantification for the assay used. ^b Upper limit of quantification for the assay used. ^c Peak, data unavailable for two FL patients. CCL, chemokine (C-C motif) ligand, CRP, C-reactive protein, CXCL, C-X-C motif chemokine ligand; FL, follicular lymphoma, GM-CSF, granulocyte-macrophage colony-stimulating factor, IFN, interferon, IL, interleukin, MCP-1, monocyte chemoattractant protein 1, POD24, disease progression <24 months after starting initial anti-CD20-containing chemoimmunotherapy, RA, receptor antagonist, SAA, serum amyloid A, TNF, tumor necrosis factor.

CD19 was detectable in 100% of 14 patients (13 FL, 1 MZL) in the broader CLINICAL TRIAL-5 population with data available at relapse after axicabtagene silolucel. Detectable CD19 was identified in all evaluable biopsies from patients on POD 24 and from patients not on POD 24. Axicabtagene silolucel product attributes were generally similar between patients on POD 24 and not on POD 24 (Table 8).

^ａ利用可能なデータに基づく：ＰＯＤ２４、ｎ＝５７；ＰＯＤ２４ではない、ｎ＝３６（ＣＣＲ７＋ＣＤ４５ＲＡ＋細胞）及びｎ＝３５（ＣＤ４／ＣＤ８比）。ａｘｉ－ｃｅｌは、アキシカブタゲンシロルユーセル、ＦＬは、濾胞性リンパ腫、ＩＦＮは、インターフェロン、ＰＯＤ２４の場合、疾患の進行は最初の抗ＣＤ２０含有化学免疫療法を開始してから２４か月未満である。

^aBased on available data: POD24, n=57; not POD24, n=36 (CCR7+CD45RA+ cells) and n=35 (CD4/CD8 ratio). axi-cel, axi-cabutagensilol-eucel, FL, follicular lymphoma, IFN, interferon, for POD24, disease progression was <24 months after starting initial anti-CD20-containing chemoimmunotherapy.

Axicabtagene siloleucel demonstrated high rates of durable responses in POD24 iNHL patients. Although median PFS was not reached in either group, the estimated PFS rate at 18 months appeared to be lower in POD24 patients than in non-POD24 patients. Among FL patients, higher pretreatment levels of analytes previously associated with relapse (CC17 [TARC] and CCL22 [MDC] (Plaks V, et al. AACR 2021.#CT036)) were observed in POD24 patients than in non-POD24 patients, potentially contributing to the difference in 18-month PFS rates. The safety profile was similarly manageable in POD24 and non-POD24 patients. Among FL patients, the posttreatment pharmacokinetic and pharmacodynamic profiles appeared to be similar in POD24 and non-POD24 patients. Axicabtagene silolucel may be a promising option for POD24 iNHL patients, especially those with high-risk disease.

Example 3
In this example, two anti-CD19 CART therapies, KTE-X19 and Axicabtageneciloreucel, were characterized. The manufacturing process of KTE-X19 was modified for Axicabtageneciloreucel to eliminate circulating lymphoma cells by positive enrichment of CD4 ⁺ /CD8 ⁺ cells. Cells were labeled with fluorescently conjugated antibodies against CD3 (pan T cell marker), CD14, CD19 (B cell marker), CD45 (pan leukocyte marker), and CD56 (activation and NK marker) and assessed by flow cytometry. Cell viability was assessed using viability dye (SYTOX, near IR) dead cell staining. The lower limit of quantification (LLOQ) of the assay was 0.2% and 5% for NK cells and monocytes. The percentage of NK cells was determined (NK cells were CD45 ⁺ , CD14 ^- , CD3 ^- , and CD56 ⁺ ; T cells were CD45 ⁺ , CD14 ^- , and CD3 ^- ). The median percentage of NK cells for 23 lots of axicabtagene ciloleucel and 97 lots of KTE-X19 was 1.9% (range, 0.8%-3.2%) and 0.1% (range, 0.0%-2.8%), respectively. The median CD3 ^- cell impurities for the same lots of axicabtagene ciloleucel and KTE-X19 were 2.4% (range, 0.9%-4.6%) and 0.5% (range, 0.3%-3.9%), respectively. KTE-X19 (Brexcavtagene Autolucel, TECARTUS) and Axicabtagene Siloleucel (YESCARTA) cell viabilities were 72% or more and 80% or more, respectively, anti-CD19 CAR expression was 24% or more and 15% or more, respectively, IFN-γ production was 190pg/mL or more and 520pg/mL or more, respectively, and the percentage of CD3 ⁺ cells was 90% or more and 85% or more. Brexcavtagene Autolucel may be composed primarily of CD3+ T cells (99.3%±0.8%), which are further classified into CD4+ (37.9%±16.5%) and CD8+ (59.3%±16.5%) subsets. The presence of circulating lymphoma cells may also play a role in the failure and depletion of anti-CD19 CAR T cells during ex vivo production.

Example 4
This example refers to 3-year results from CLINICAL TRIAL-4 (NCT02625480), a Phase 1/2 multicenter study evaluating the safety and efficacy of KTE-X19, an autologous anti-CD19 chimeric antigen receptor (CAR) T therapy (described above), in pediatric/adolescent patients with relapsed/refractory (R/R) B-cell acute lymphoblastic leukemia (B-ALL; median follow-up for all treated patients: 36.1 months). Two formulations were investigated in Phase 1, one with a total volume of 40 mL and the other with a volume of 68 mL, for patients receiving a lower dose of 1×10 ⁶ CAR T cells/kg. The 40 mL formulation was intended to maintain cell density and cell viability during the freeze/thaw process. Subjects weighing >100 kg received a maximum fixed dose of 2×10 ⁸ or 1×10 ⁸ anti-CD19 CAR T cells. The primary endpoint was the incidence of dose-limiting toxicity (DLT). Of 31 enrolled patients, 24 (median age 13.5 years, range 3-20) received KTE-X19 with a median follow-up of 36.1 months. No DLTs were observed. All treated patients had grade 3 or higher adverse events, typically hypotension (50%) and anemia (42%). The rates of grade 3 cytokine release syndrome were 33%, 75%, 27%, and 22% across all treatment arms at ^2x106 , ^1x106 (68 mL formulation), and ^1x106 (40 mL formulation) CAR T cells/kg, with 21%, 25%, 27%, and 11% of patients, respectively, experiencing grade 3 or higher neurological events. Overall complete remission (CR) rates (CR and CR with incomplete hematologic recovery) were ⁶⁷ %, 75%, 64%, and 67% in all treatment arms at 2x106, ^1x106 (68 mL formulation), and ^1x106 (40 mL) CAR T cells/kg, respectively. Overall MRD negativity was 100% among responders. Median duration of remission and overall survival were not reached in the ^1x106 (40 mL) arm (recommended Phase 2 dose). Pediatric/adolescent patients with R/R B-ALL achieved high MRD-negative remission rates with a manageable safety profile after a single dose of KTE-X19. Phase 2 is ongoing at the ^1x106 CAR T cells/kg (40 mL) dose.

In the Phase 1 portion of CLINICAL TRIAL-4, eligible patients were defined as age 21 years or younger, weight ≥6 kg, with R/R B-ALL only, ineligible for first-line therapy, R/R after ≥2 systemic therapies, or R/R after alloSCT if patients were ≥100 days from alloSCT at enrollment and had not received immunosuppressants for ≥4 weeks prior to enrollment. Prior treatment with blinatumomab was permitted. Dose-limiting toxicities (DLTs) were defined as follows: grade 4 hematologic toxicity lasting >30 days (except lymphopenia) if not attributable to underlying disease; any KTE-X19-related grade 3 non-hematologic toxicity lasting >7 days; any KTE-X19-related grade 4 non-hematologic toxicity regardless of duration (exceptions are listed in Table 9). Specific bridging chemotherapy after leukapheresis was permitted and completed in at least 7 days or 5 half-lives, whichever was shorter, before initiating conditioning chemotherapy consisting of fludarabine 25 mg/ ^m2 /day intravenously (IV) on days -4, -3, and -2, and a single dose of cyclophosphamide 900 mg/ ^m2 IV on day -2. A single IV infusion of KTE-X19 was administered on day 0 at a target dose of ^2x106 or ^1x106 CAR T cells/kg. A minimum of 7 days of hospitalization was required after infusion. All patients who completed the 3-month visit proceeded to a long-term follow-up period for survival and disease status, followed every 3 months until month 18, every 6 months from months 24 to 60, and then annually until 15 years of age. Patients could be removed from the study if they withdrew consent for further follow-up, were lost to follow-up, or died. Allogeneic stem cell transplantation (alloSCT) was not required per protocol but was allowed at the investigator's discretion.

Additional Phase 1 Inclusion Criteria Morphologic disease with >5% myeloblasts:
1. Lansky or Karnofsky performance status ≥ 80% at screening 2. Patients with Philadelphia chromosome positive disease were also eligible if they were intolerant to tyrosine kinase inhibitor therapy or had R/R disease despite treatment with at least two different tyrosine kinase inhibitors (TKIs) 3. For patients previously treated with blinatumomab, leukemic blasts with CD19 expression ≥ 90% were required 4. Absolute neutrophil count (ANC) ≥ 500/μL if, in the investigator's opinion, cytopenia is due to underlying leukemia and not potentially reversible with treatment of the leukemia
5. Platelet count ≥ 50,000/μL if, in the investigator's opinion, the cytopenia is due to underlying leukemia and is not potentially reversible with treatment of the leukemia. 6. Absolute lymphocyte count ≥ 100/μL. 7. Adequate renal, hepatic, pulmonary, and cardiac function defined as:
a. Creatinine clearance of 60 mL/min or more (estimated by Cockcroft-Gault or Schwartz formula)
b. Serum alanine aminotransferase and aspartate aminotransferase less than or equal to 5 x the upper limit of normal (ULN); c. Total bilirubin less than or equal to 1.5 x the ULN (except in patients with Gilbert's syndrome).
d. Left ventricular fractional shortening ≥ 30% or left ventricular ejection fraction ≥ 50%, no evidence of pericardial effusion as determined by echocardiography, and no clinically significant arrhythmias; e. Absence of clinically significant pleural effusion; f. Baseline oxygen saturation on room air > 92%. 8. Females of childbearing potential (defined as having menarche) must have a negative serum or urine pregnancy test.

Additional Phase 1 Exclusion Criteria 1. Diagnosis of Burkitt's leukemia/lymphoma according to the World Health Organization classification, or blast crisis of chronic myeloid leukemia 2. History of malignancy other than non-melanoma skin cancer or carcinoma in situ unless disease-free for ≥3 years 3. History of severe hypersensitivity reaction to aminoglycosides or any of the drugs used in this study 4. History or current history of any central nervous system (CNS) disease, e.g., seizure disorder, cerebrovascular ischemia/hemorrhage, dementia, cerebellar disease, any autoimmune disease involving the CNS, reversible leukoencephalopathy, or cerebral edema a. Detectable cerebrospinal fluid blasts in cerebrospinal fluid samples with <5 leukocytes/mm3 with neurological changes (CNS-2) or detectable cerebrospinal fluid blasts in cerebrospinal fluid samples with ≥5 leukocytes/mm3 with or without neurological changes (CNS-3) were also excluded 5. 5. History of concomitant genetic syndromes associated with bone marrow failure, such as Fanconi anemia, Kostmann syndrome, or Schwachman-Diamond syndrome. 6. History of myocardial infarction, cardiac angioplasty or stent placement, unstable angina, or other clinically significant cardiac disease within 12 months of enrollment. 7. History of symptomatic deep vein thrombosis or pulmonary embolism within 6 months of enrollment. 8. Primary immunodeficiency. 9. Known infection with HIV, Hepatitis B, or Hepatitis C virus. A history of Hepatitis B or Hepatitis C is permitted if the viral load is undetectable by quantitative polymerase chain reaction (PCR) and/or nucleic acid testing. 10. Current history of fungal, bacterial, viral, or other infections that are uncontrolled or require intravenous administration of antimicrobial agents for management. Uncomplicated urinary tract infections and uncomplicated bacterial pharyngitis are permitted after consultation with a Kite Medical Monitor if they have responded to active treatment. 11. Prior Medications a. Salvage systemic therapy (including chemotherapy, Ph+ALL TKI, and blinatumomab) within 1 week or 5 half-lives (whichever is shorter) prior to enrollment
b. Prior CD19-targeted therapy other than blinatumomab c. History of Common Terminology Criteria for Adverse Events grade 4 neurological event or grade 4 CRS due to prior CD19-targeted therapy (Lee et al 2014)
d. Alemtuzumab within 6 months prior to enrollment, clofarabine or cladribine within 3 months prior to enrollment, or PEG-asparaginase within 3 weeks prior to enrollment e. Donor lymphocyte infusion within 28 days prior to enrollment f. Any medication used for graft-versus-host disease (GVHD) within 4 weeks prior to enrollment (e.g., calcineurin inhibitors, methotrexate, mycophenolate, rapamycin, thalidomide) or immunosuppressive antibodies used within 4 weeks prior to enrollment (e.g., anti-CD20, anti-tumor necrosis factor, anti-interleukin 6, or anti-interleukin 6 receptor).
g. At least 3 half-lives must have elapsed since any prior systemic inhibitory/stimulatory immune checkpoint molecule therapy (e.g., ipilimumab, nivolumab, pembrolizumab, atezolizumab, OX40 agonists, 4-1BB agonists)
h. Pharmacological dose corticosteroid therapy (≥0.7 mg/kg/day hydrocortisone or equivalent dose corticosteroids) and other immunosuppressants must be avoided for 7 days prior to enrollment 12. Presence of any indwelling lines or drains (e.g., percutaneous nephrostomy tube, indwelling Foley catheter, biliary drain, or pleural/peritoneal/pericardial catheter). Ommaya reservoirs and dedicated central venous access catheters, e.g., Port-a-Cath or Hickman catheters, are permitted 13. Acute GVHD grade II-IV according to the Glucksberg criteria or severity B-D according to the International Bone Marrow Transplant Registry index. Acute or chronic GVHD requiring systemic treatment within 4 weeks prior to enrollment
14. Live vaccine within 4 weeks prior to enrollment 15. Pregnant or breastfeeding women of childbearing potential due to potential adverse effects of preparatory chemotherapy on the fetus or infant 16. Male and female subjects of childbearing potential who are not willing to practice contraception from the time of consent until 6 months after completion of KTE-X19 17. Subjects who, in the opinion of the investigator, are unlikely to complete all study visits or procedures required by the protocol, including follow-up visits, or who are unlikely to comply with study requirements for participation 18. History of autoimmune disease (e.g., Crohn's disease, rheumatoid arthritis, systemic lupus) within the last 2 years resulting in end organ damage or requiring systemic immunosuppression/systemic disease-modifying agents.

The study design and treatment were as follows: The objective of Phase 1 was to evaluate the safety of KTE-X19 and to determine a recommended Phase 2 dose (RP2D) of KTE-X19 based on the incidence of dose-limiting toxicities (DLTs) and the overall safety profile. DLTs were defined as follows: grade 4 hematological toxicity (excluding lymphopenia) lasting >30 days, if not attributable to underlying disease, any KTE-X19-related grade 3 non-hematologic toxicity lasting >7 days; any KTE-X19-related grade 4 non-hematologic toxicity regardless of duration (exceptions are listed in Table 9). Specific bridging chemotherapy after leukapheresis was permitted and completed in at least 7 days or 5 half-lives, whichever was shorter, before initiating conditioning chemotherapy consisting of fludarabine 25 mg/m2/day intravenously (IV) on days ^-4 , -3, and -2, and a single dose of cyclophosphamide 900 mg/m2 IV on day -2. A single IV infusion of KTE-X19 was administered on day 0 at a target dose of ^2x106 or ^1x106 CAR T cells/kg. A minimum of 7 days of hospitalization was required after infusion. All patients who completed the 3-month visit proceeded to a long-term follow-up period for survival and disease status every 3 months until 18 months, every 6 months from 24 to 60 months, and then annually until 15 years. Patients could be removed from the study if they withdrew consent for further follow-up, were lost to follow-up, or died. Allogeneic stem cell transplantation (alloSCT) was not required per protocol but was allowed at the investigator's discretion.

ＣＲＳは、サイトカイン放出症候群、ＤＬＴ、用量制限的毒性；ＴＬＳは、腫瘍溶解症候群を意味する。

CRS means cytokine release syndrome; DLT, dose-limiting toxicity; TLS, tumor lysis syndrome.

DLT was evaluated in the first three patients treated with a starting dose of ^2x106 CAR T cells/kg. One additional patient was enrolled and administered ^2x106 CAR T cells/kg. The safety review team followed these patients for 28 days after infusion and evaluated safety data after subsequent patients were administered ^1x106 CAR T cells/kg to evaluate the possibility of reducing the risk of CRS and NE to improve the risk:benefit ratio (Shah BD et al., J Clin Oncol 37:abstr 7006,2019; Shah BD et al., Blood In Press,2021). To further optimize the risk:benefit ratio, the dosage formulation was modified from 68 mL to 40 mL for the second cohort of patients at ^1x106 CAR T cells/kg. Given the expected patient weight in this pediatric study population, the 40 mL formulation was intended to provide a higher cell density than the 68 mL formulation to mitigate the potential risk of a lower final product volume. Patients underwent leukapheresis to obtain cells for CAR T cell manufacturing, followed by subsequent conditioning chemotherapy. Fresh leukapheresis material was used for CAR T cell manufacturing. Specific bridging chemotherapy was allowed between leukapheresis and conditioning chemotherapy (Table 10).

^＊上記レジメンのいずれかと組み合わせたＴＫＩの使用は、Ｐｈ＋ＡＬＬ患者及びＰｈ様ＡＬＬ患者に対して許可される；^†ビンクリスチンに耐えられない患者については、別のアルカロイドが使用され得る。^‡ＦＬＡＧ化学療法投与中のメトトレキサートの髄腔内投与との併用治療は、回避するべきである。ＡＬＬは、急性リンパ芽球性白血病、ＡＮＣは、絶対好中球数、ＦＬＡＧ、フルダラビン、高用量シタラビン、及びＧ－ＣＳＦ；Ｇ－ＣＳＦは、顆粒球コロニー刺激因子、ＩＶは、静脈内、Ｐｈは、フィラデルフィア染色体、ＰＯ、経口；ＳＣ、皮下；ＴＫＩは、チロシンキナーゼ阻害剤、ＶＡＤ、ビンクリスチン、ドキソルビシン、及びデキサメタゾン。

^* The use of a TKI in combination with any of the above regimens is permitted for patients with Ph+ ALL and Ph-like ALL; ^† For patients who cannot tolerate vincristine, another alkaloid may be used. ^‡ Concomitant treatment with intrathecal methotrexate during FLAG chemotherapy should be avoided. ALL, acute lymphoblastic leukemia; ANC, absolute neutrophil count, FLAG, fludarabine, high-dose cytarabine, and G-CSF; G-CSF, granulocyte colony-stimulating factor, IV, intravenous; Ph, Philadelphia chromosome, PO, oral; SC, subcutaneous; TKI, tyrosine kinase inhibitor, VAD, vincristine, doxorubicin, and dexamethasone.

KTE-X19 was administered on day 0 at a target dose of ^2x106 or ^1x106 CAR T cells/kg (68 mL or 40 mL formulation), respectively. A minimum of 7 days of hospitalization after infusion was required, with response assessments at predefined time points thereafter. All patients who completed the 3-month visit proceeded to a long-term follow-up period for survival and disease status, followed every 3 months until 18 months, every 6 months from 24 to 60 months, and then annually until 15 years. Patients could be removed from the study if they withdrew consent for further follow-up, were lost to follow-up, or died. Allogeneic stem cell transplantation (alloSCT) was not required per protocol, but was allowed at the investigator's discretion.

Patients receiving 1×10 ⁶ CAR T cells/kg (40 mL) were treated under the revised toxicity management guidelines. Tocilizumab was only administered for neurological events (NE) in the setting of cytokine release syndrome (CRS), and steroids were initiated for grade 2 NE. For the 1×10 ⁶ CAR T cells/kg (68 mL) cohort, steroids were initiated for grade 3 NE under the original toxicity management guidelines (Table 11).

^＊予防的レベチラセタムは全てのグレードに適用される。^†代替療法の開始は、メディカルモニターと協議するべきであり、アナキンラ、シルツキシマブ、ルキソリチニブ、シクロホスファミド、静脈内免疫グロブリン、及び抗胸腺細胞グロブリンを含む（ただし、これらに限定されない）。
ＢＩＤは、１日２回、ＣＲＳは、サイトカイン放出症候群、ＣＳＦは、脳脊髄液、ＥＥＧは、脳電図、ＩＣＵは、集中治療室、ＩＶは、静脈内、ＭＲＩは、磁気共鳴画像検を意味する

^* Prophylactic levetiracetam is indicated for all grades. ^† Initiation of alternative therapies should be discussed with a medical monitor and include (but are not limited to) anakinra, siltuximab, ruxolitinib, cyclophosphamide, intravenous immunoglobulin, and antithymocyte globulin.
BID means twice a day, CRS means cytokine release syndrome, CSF means cerebrospinal fluid, EEG means electroencephalogram, ICU means intensive care unit, IV means intravenous, MRI means magnetic resonance imaging.

Results and Evaluation The primary endpoint of Phase 1 was the incidence of DLT in the DLT-evaluable set, which included the first three patients treated with KTE-X19 at a dose of ^2x106 CAR T cells/kg. Secondary endpoints included safety, overall CR rate (CR+CRi), duration of remission (DOR), MRD negativity rate, alloSCT rate, OS, and relapse-free survival (RFS). Adverse events, including individual symptoms of cytokine release syndrome (CRS) and neurological events (NE), were graded according to the NCI Common Terminology Criteria for Adverse Events (AEs) version 4.03. CRS was graded according to the modified criteria of Lee et al., Blood. 2014;124(2):188-195. Overall response was determined by the investigator after bone marrow and peripheral blood evaluation as detailed in the table below (Cheson BD et al., J Clin Oncol. 2007;25(5):579-586). Bone marrow and response evaluations were performed on day 28 and at months 2 and 3. Patients who received bridging chemotherapy required additional bone marrow aspirates between the end of bridging chemotherapy and day -4 (+/- 2 days). For patients with extramedullary disease, response was assessed according to the revised International Working Group criteria for lymphoma for extramedullary and CNS disease response criteria as detailed in the table below (Cheson BD et al., J Clin Oncol. 2007;25(5):579-586). Minimal residual disease (MRD) was tested by flow cytometry (Neogenomics) with a sensitivity of 0.01% using the following markers: CD3, CD9, CD10, CD13/CD33, CD19, CD20, CD34, CD38, CD45, CD58, and CD71 (Gupta S et al., Leukemia. 2018; 32(6): 1370-1379; Borowitz MJ et al., Blood. 2015; 126(8): 964-971; Bruggemann M et al., Hematology Am Soc Hematol Educ Program. 2017; 2017(1): 13-21). MRD negativity is defined as MRD < ^10-4 by standard assessment. Aliquots of bone marrow aspirates taken at day 28 and at months 2 and 3 were analyzed for MRD. Translational analyses were performed on product, blood, and tumor samples to evaluate the pharmacokinetic and pharmacodynamic profile of KTE-X19 in pediatric R/R B-ALL as an exploratory endpoint. Pharmacokinetic and pharmacodynamic evaluations and associations with clinical outcomes have been previously described (Locke FL et al., Mol Ther. 2017;25(1):285-295). Overall disease response was as described in Table 12.

^＊Ｐｌｔ及びＡＮＣの単位は１μＬ当たりである。ＡＮＣ及びＰｌｔ値は、ＢＭ評価が実行されるたびに評価されるべきである。行われない場合、奏功評価に使用されるＡＮＣ及びＰｌｔ値は、ＢＭ結果の７日前の任意の時間からＢＭ結果後の任意の時間までであり得る。^§非ＣＮＳ ＥＭＤについて評価された患者において、全体的な疾患奏功の評価に使用される画像化及び骨髄の結果は、互いに３０日以内でなければならない。^‡ＢＭにおける形態による芽球。^＃２８日目又は最初の推定奏功時のいずれか早い方における、ＣＲに達した患者について。^＊＊循環白血病は、形態による１％未満の循環芽球ではない。循環白血病は、形態による１％以上の循環芽球である。形態による芽球が１％以上であり、白血病の他の証拠がない場合、芽球が白血病であることを確認するために、フロー又は分子研究を行うべきである。ＡＮＣは、好中球絶対数、ＢＭ、骨髄；ＣＮＳ、中枢神経系；ＣＲ、完全寛解；ＣＲｈは、部分的な血液学的回復を伴う完全寛解、ＣＲｉは、不完全な血液学的回復を伴う完全寛解、ＥＭＤは、骨髄外疾患；ＰＤは、進行性疾患、Ｐｌｔは、血小板；ＰＲは、部分奏効を意味する。

^* Units of Plt and ANC are per μL. ANC and Plt values should be assessed each time a BM evaluation is performed. If not, ANC and Plt values used for response evaluation can be any time from 7 days prior to the BM result to any time after the BM result. ^§ In patients evaluated for non-CNS EMD, imaging and bone marrow results used to evaluate overall disease response must be within 30 days of each other. ^‡ Blasts by morphology in BM. ^# For patients who achieve CR at day 28 or time of first estimated response, whichever occurs first. ^** Circulating leukemia is not <1% circulating blasts by morphology. Circulating leukemia is ≥1% circulating blasts by morphology. If ≥1% blasts by morphology and there is no other evidence of leukemia, flow or molecular studies should be performed to confirm that the blasts are leukemia. ANC, absolute neutrophil count; BM, bone marrow; CNS, central nervous system; CR, complete remission; CRh, complete remission with partial hematologic recovery, CRi, complete remission with incomplete hematologic recovery, EMD, extramedullary disease; PD, progressive disease, Plt, platelets; PR, partial response.

Results Patients Thirty-one patients were enrolled and underwent leukapheresis between February 17, 2016 and August 1, 2018. The median time from leukapheresis to KTE-X19 product release was 14.0 days (range, 9.0-20.0) for all treated patients, 16.5 days (range, 12.0-23.0) from leukapheresis to delivery to study site, and 27.0 days (range, 18.0-41.0) from leukapheresis to infusion. Data cut-off date was September 9, 2020. Of the 31 enrolled patients, 24 (77%) received conditioning chemotherapy and were subsequently dosed. Seven patients were not dosed for the following reasons: adverse events (AEs; n=1), product unavailability (n=3), ineligible due to AEs (n=1), product unavailability and ineligible (n=1), and death (n=1). Twenty-four patients received conditioning chemotherapy followed by KTE-X19. Four patients received the ^2x106 CAR T cells/kg dose, 11 patients received the 1x106 CAR T cells/kg (68 mL) dose formulation, and ⁹ received the ^1x106 CAR T cells/kg (40 mL) dose formulation. The median follow-up of all treated patients was 36.1 months (range, 24.0-53.9). The median age of treated patients was 13.5 years (range, 3-20), 42% of patients had received 3 or more prior lines of therapy, 29% had first-line refractory disease, 25% were R/R after alloSCT, and median bone marrow blasts at screening was 44% (range, 6-99; Table 13). Prior to enrollment, 6 (25%) patients had received up-front allo-SCT, 8 (33%) had received up-front blinatumomab, of which 3 (13%) had received blinatumomab as their last prior therapy, and 1 (4%) had extramedullary disease. Of the 31 enrolled patients, 30 (97%) received bridging therapy per protocol, with a new baseline disease assessment performed immediately prior to lymphodepleting chemotherapy. Safety and efficacy analyses are presented for all 24 patients who received the drug.

Safety Among the 3 DLT-evaluable patients who received ^2x106 CAR T cells/kg, no DLTs were observed. All treated patients (n=24) experienced at least one Grade ≥ 3 AE, most commonly hypotension (50%) and anemia (42%, Table 14). Serious AEs of any grade occurred in 71% of patients. Grade ≥ 3 infections occurred in 42% of patients.

^＊表には、全患者の２０％以上で生じた、任意のグレードの有害事象が含まれる。

^* Table includes adverse events of any grade occurring in ≥20% of all patients.

In all treated patients (n=24), CRS was reported in 21 patients (88%), with 8 patients (33%) experiencing grade 3 or higher CRS according to the modified Lee grading criteria (Table 15) (Lee DW et al., Blood 124:188-95, 2014). No grade 4 or grade 5 CRS events occurred. The most common grade 3 or higher CRS symptoms were hypotension (50%) and fever (25%). Any grade and grade 3 or higher hypoxia were observed in 13% and 8% of patients, respectively. The median time to onset of CRS and duration after KTE-X19 infusion were 5 days (range, 1-14) and 7 days, respectively, and all events resolved.

^＊全患者の１０％以上で生じるＣＲＳ症状及び神経学的事象を含む。^†サイトカイン放出症候群は、Ｌｅｅ ｅｔ ａｌ．Ｂｌｏｏｄ，２０１４によって提案された修正版グレーディングシステムに従って分類された。

^* Includes CRS symptoms and neurological events occurring in 10% or more of all patients. ^† Cytokine release syndrome was classified according to the modified grading system proposed by Lee et al. Blood, 2014.

Among all treated patients, NE of any grade was reported in 16 patients (67%), with grade ≥ 3 events occurring in 5 patients (21%), with encephalopathy (13%) being the most common grade ≥ 3 event (Table 15). One fully reversible grade 4 NE occurred (cerebral edema) in a patient receiving a dose of 1 x 106 CAR T cells/kg (68 mL). For this event, the patient was treated with dexamethasone, mannitol, sodium chloride, and tocilizumab. There were no grade 5 NE. Overall, the median time to onset of NE was 9.5 days (range, 3-60) after infusion, and the median duration of NE was 8 days. NE resolved in 14 of 16 patients (88%). The NE in the remaining 2 patients was ongoing at the time of death due to AE (n=1) or progressive disease (n=1).

Of all treated patients, 42% received steroids, 63% received tocilizumab, and 46% received vasopressors (Table 15). Improved overall safety was observed in the nine patients treated with a dose of 1×10 ⁶ CAR T cells/kg (40 mL) under the revised toxicity management compared with the four patients treated with 2×10 ⁶ CAR T cells/kg and the 11 patients treated with 1×10 ⁶ CAR T cells/kg (68 mL) under the original guidelines. Of patients receiving 2×10 ⁶ CAR T cells/kg, 75% experienced grade 3 or higher CRS compared with 27% and 22% receiving 1×10 ⁶ CAR T cells/kg (68 mL and 40 mL, respectively). Grade ≥ 3 NE was observed in 25% of patients receiving ^2x106 CAR T cells/kg and 27% of patients receiving ^1x106 CAR T cells/kg (68 mL), but was lowest (11%) in patients receiving ^1x106 CAR T cells/kg (40 mL). In addition, the median time to onset of NE and CRS appeared to be delayed in the ^1x106 CAR T cells/kg dose cohort compared to the ^2x106 CAR T cells/kg dose cohort.

Of the 8 patients (33%) who died on study, 6 died from progressive disease (median 190.5 days post-KTE-X19 infusion) and 2 patients died from AEs considered unrelated to KTE-X19 (non-grade 5 B-ALL), including disseminated mucormycosis (n=1, 15 days post-KTE-X19 infusion) and E. coli sepsis (n=1, 409 days post-KTE-X19 infusion). Of the patients who died, 3 patients received ^2x106 CAR T cells/kg, 4 received ^1x106 CAR T cells/kg (68 mL), and 1 received ^1x106 CAR T cells/kg (40 mL). No patients tested positive for replicating retrovirus or antibodies to the anti-CD19 CAR at any time point.

Efficacy At a median follow-up of 36.1 months (range, 24.0-53.9), all treated patients (n=24) were evaluable for efficacy. The investigator-assessed overall remission rate was 67%, with 29% of patients (n=7) achieving CR and 38% achieving CRi (n=9; Table 16). In the ^2x106 , ^1x106 (68 mL), and ^1x106 (40 mL) CAR T cells/kg dose groups, the CR+CRi rates were 75%, 64%, and 67%, respectively. The median time from infusion to CR+CRi across dose levels was 30 days (range, 26-113 days). The overall MRD negativity rate was 100% among the 16 patients with CR+CRi. Overall, ¹⁶ patients (67%) underwent alloSCT as subsequent consolidation therapy, including 2, 8, and 6 patients in the 2x106, ^1x106 (68 mL), and ^1x106 (40 mL) CAR T cells/kg dose groups, respectively. Fourteen of the 16 patients (88%) who achieved CR+CRi (2, ⁷ , and 5 patients in the 2x106, ^1x106 [68 mL], and ^1x106 [40 mL] CAR T cells/kg dose groups, respectively) underwent subsequent alloSCT. Two of these patients relapsed prior to subsequent alloSCT, and both received consolidation chemotherapy before proceeding to alloSCT. Of the two patients who achieved CR+CRi but did not undergo subsequent alloSCT, one died of progressive disease and one was lost to follow-up. Two patients who did not achieve any response proceeded to subsequent alloSCT and achieved a CR as their response to alloSCT. The median time to transplant for all treated patients was 2.3 months (range, 1.4-24.9) after KTE-X19.

The median DOR for the 16 patients who achieved CR+CRi after KTE-X19 was 7.2 months (95% CI, 4.1-14.2) after censoring of subsequent alloSCT and was not reached at 4.1 and 10.7 months in the ^2x106 , ^1x106 (68 mL), and ^1x106 (40 mL) CAR T cells/kg dose groups, respectively. The median DOR was 14.2 months (95% CI, 3.9-NE) with no subsequent censoring of alloSCT and reinitiation of tyrosine kinase inhibitors. The median DOR among the 14 patients with CR+CRi who received subsequent alloSCT after KTE-X19 was 10.7 months (95% CI, 7.2-14.2). Median RFS for all treated patients (n=24) was 5.2 months (95% CI, 0.0-17.8). Median RFS for the 1×10 ⁶ CAR T cells/kg (40 mL) group was not reached, being 5.2 months (95% CI, 0.0-5.2) and 9.1 months (95% CI, 0.0-17.8) for the 2×10 ⁶ and 1×10 ⁶ (68 mL) cells/kg cohorts, respectively. Median RFS among the 16 patients who proceeded to subsequent alloSCT was 9.1 months (95% CI, 9.1-17.8). Median OS was not reached among all treated patients and in the 1×10 ⁶ CAR T cells/kg dose group, being 8.0 months for the 2×10 ⁶ CAR T cells/kg dose group. The 24-month OS rate for the 1 x ¹⁰⁶ cells/kg (40 mL) dose was 87.5% (95% CI, 38.7-98.1) for the 1 x ¹⁰⁶ cells/kg (40 mL) dose and 72.7% (95% CI, 37.1-90.3) for the 1 x ¹⁰⁶ cells/kg (68 mL) dose. Overall, as of the data cut-off date, 33% (8/24) of treated patients had died, one patient had discontinued due to withdrawal of full consent, and one was lost to follow-up. The remaining 58% (14/24) of patients were still alive and under continued follow-up as of the data cut-off date, all of whom underwent subsequent alloSCT after KTE-X19. Based on safety and efficacy data analysis, the RP2D was 1 x ¹⁰⁶ KTE-X19 cells/kg (40 mL formulation) with revised toxicity management.

Translational Analysis CAR T cell proliferation in peripheral blood, measured by polymerase chain reaction (PCR) and expressed as CAR gene copies/μg DNA in blood, was observed across dose groups, reaching peak CAR T cell levels by day 14, followed by CAR T cell shrinkage to baseline (Table 17). Median CAR T cell levels were undetectable in blood by PCR across all dose groups at 3 months after KTE-X19 infusion (Table 17). Median peak CAR gene copies/μg DNA in blood was similar between the 1×10 ⁶ CAR T cells/kg dose cohorts, but higher in the 2×10 ⁶ CAR T cells/kg cohort (FIG. 10A). Patients who achieved CR+CRi trended toward higher peak blood CAR gene copies/μg DNA in blood than non-responders, as did MRD-negative patients compared to MRD-positive (FIG. 10C). Blood CAR gene copies/μg DNA tended to be higher in patients with grade 3 or higher NE compared with patients with grade 2 or lower NE (FIG. 10D). However, with this limited sample size, there was no clear difference in peak blood CAR gene copies/μg DNA in patients with either high- or low-grade CRS. The median peak blood CAR gene copies/μg DNA was 5.16×10 ⁴ (range, 0-2.40×10 ⁵ ) in 16 patients who did not receive prior blinatumomab and 6.15×10 ³ (range, 0-2×49×10 ⁵ ) in 8 patients who did receive prior blinatumomab.

Peak levels of multiple key serum cytokines, chemokines, and pro-inflammatory biomarkers occurred by day 7. Proportional to peak CAR expansion, several serum analytes tended to be higher in patients receiving 2×10 ⁶ CAR T cells/kg compared to 1×10 ⁶ CAR T cells/kg (interleukin [IL]-2, IL-5, IL-6, IL-8, IL-10, IL-15, IL-16, ferritin, granzyme B, intercellular adhesion molecule 1 [ICAM-1], interferon gamma [IFN-γ], and tumor necrosis factor alpha [TNF-α]) ( FIG. 11 ; Table 18).

Peak serum levels of the analytes VCAM-1 and IL-16 were associated with grade 3 or higher CRS. Such associations were not observed in subjects with NE 3 or higher. This may have been due to the small number of patients with NE 3 or higher (Table 19).

^＊値は、使用したアッセイにおける定量化の下限を表す。^†値は、使用したアッセイにおける定量化の上限を表す。ＣＲＰは、Ｃ反応性タンパク質、ＣＸＣＬ、Ｃ－Ｘ－Ｃモチーフケモカインリガンド；ＧＭ－ＣＳＦは、顆粒球マクロファージコロニー刺激因子、ＩＦＮγは、インターフェロンγ、ＩＣＡＭは、細胞間接着分子、ＩＬは、インターロイキン、ＩＰ、インターフェロンγ誘導タンパク質；ＭＣＰ、単球誘引性タンパク質；Ｒα、受容体α；ＲＡは、受容体アンタゴニスト、ＳＡＡは、血清アミロイドＡ、ＴＮＦ－αは、腫瘍壊死因子α、ＶＣＡＭは、血管細胞接着分子を示す。

^* Values represent the lower limit of quantification for the assay used. ^† Values represent the upper limit of quantification for the assay used. CRP, C-reactive protein; CXCL, C-X-C motif chemokine ligand; GM-CSF, granulocyte-macrophage colony-stimulating factor; IFNγ, interferon gamma; ICAM, intercellular adhesion molecule; IL, interleukin; IP, interferon gamma-inducible protein; MCP, monocyte-attractant protein; Rα, receptor alpha; RA, receptor antagonist; SAA, serum amyloid A; TNF-α, tumor necrosis factor alpha; VCAM, vascular cell adhesion molecule.

Product characteristics were similar across dose levels (Table 20). Levels of less differentiated CCR7+ T cells in the product were higher in CR+CRi patients and tended to be higher in MRD-negative patients (Table 21).

This product profile also appeared to be associated with higher levels of neurotoxicity, but was not associated with CRS. The ratio of CD4 to CD8 T cells was not associated with response or toxicity.

ＣＲ、完全寛解；ＣＲｉは、不完全な血液学的回復を伴う完全寛解、ＣＲＳは、サイトカイン放出症候群、ＭＲＤは、微小残存病変、ＮＥは、神経学的事象を示す。

CR, complete remission; CRi, complete remission with incomplete hematologic recovery, CRS, cytokine release syndrome, MRD, minimal residual disease, NE, neurological event.

In Phase 1 of CLINICAL TRIAL-4, no DLTs were observed with KTE-X19 among DLT-evaluable pediatric or adolescent patients with R/R B-ALL. No DLTs were observed with the initial dose of ^2x106 CAR T cells/kg, but to further improve the risk:benefit ratio, a lower dose of ^1x106 CAR T cells/kg with a 68 mL formulation was explored in a second cohort of patients, and dosing and toxicity management was further optimized with a 40 mL formulation and revised toxicity management in a third cohort of ^1x106 CAR T cells/kg. This resulted in a more optimal risk:benefit ratio for the ^1x106 CAR T cells/kg (40 mL) dose level, with notable improvements in CRS and NE. In addition, the MRD negativity rate was 73% or higher for all formulations, but the MRD negativity rate and CR-only rate were highest in patients receiving ^1x106 CAR T cells/kg (40 mL).After 36.1 months of follow-up in all treated patients, the ^1x106 CAR T cells/kg (40 mL) cohort still did not reach median DOR, RFS, and OS, with a 24-month OS rate of 87.5%, potentially suggesting significant durability of response with optimized KTE-X19 dosing/formulation in pediatric/adolescent patients with R/R B-ALL.

The role of alloSCT following anti-CD19 CAR T-cell therapy in pediatric/adolescent patients with R/R B-ALL is still poorly understood, and studies in adult populations have provided somewhat conflicting results (Park JH et al., N Engl J Med 378:449-459, 2018; Hay KA et al., Blood 133:1652-1663, 2019). In this study, after a median follow-up of 36.1 months (median for all treated patients), patients treated with RP2D of ^1x106 CAR T cells/kg (40 mL) did not reach median DOR and OS censored with subsequent alloSCT. Fourteen of 16 patients (88%) who achieved CR+CRi, including five treated with RP2D, received alloSCT as subsequent treatment. AlloSCT was not required according to the protocol but was allowed at the investigator's discretion. Although CLINICAL TRIAL-4 was not designed to evaluate outcomes after subsequent treatment, given that most responding patients did not proceed to alloSCT after KTE-X 19, evaluation of DOR without censoring of subsequent treatment, including alloSCT, revealed a favorable median value of 14.2 months. In addition, the median RFS with censoring of subsequent alloSCT was 5.2 months, whereas the uncensored value was 9.1 months, indicating a potential favorable impact of alloSCT after KTE-X 19. It has previously been reported that children and young adults with R/R CD19+ ALL who have not received alloSCT but who receive consolidation alloSCT after anti-CD19 CAR T-cell therapy trend toward improved leukemia-free survival with 1 year or more of follow-up (Summers C. et al., Blood 132:967-967, 2018). In a recently published phase 1 study of anti-CD19 CAR T-cell therapy in children and young adults with R/R B-ALL, in which 75% of MRD-negative responders proceeded to alloSCT, the median OS at 4.8 years of follow-up was 70.2 months after alloSCT, the 5-year event-free survival rate after alloSCT was 61.9%, and the cumulative recurrence rate after alloSCT was only 9.5% (Shah NN. et al., Journal of Clinical Oncology 0:JCO.20.02262). These data suggest that subsequent alloSCT may be important for maintaining remission after CAR T-cell therapy in pediatric R/R B-ALL. A retrospective review in pediatric and young adult patients found that CD34-selected T cell-depleted alloSCT after CAR T cell therapy could result in improved transplant-related mortality and OS compared to unmodified alloSCT (Fabrizio VA et al., Bone Marrow Transplant 55:2160-2169, 2020). Given that median blood CAR T cell levels in CLINICAL TRIAL-4 were undetectable across all doses at 3 months post-infusion and median time to alloSCT was 2.3 months, and given the small overall patient numbers and high rate of subsequent alloSCT, the association between CAR T cell persistence and durability of response should be explored. In contrast to the present study, studies using tisagenlecleucel performed subsequent alloSCT in a minority of responding patients (12%-13%) with a short median follow-up of 13.1 months, while approximately 40% of responding patients who received tisagenlecleucel had already relapsed, mostly with CD19-negative leukemia, despite persistence of CAR T cells (Maude SL et al, N Engl J Med 378:439-448,2018; Grupp SA et al, Blood 132:895-895,2018; Pasquini MC et al., Blood Adv 4:5414-5424,2020.).

While differences in study design and patient populations prevent direct inter-study comparisons, a recent study with blinatumomab, which also targets CD19, shows a median OS of only 7.7 months in pediatric R/R B-ALL, similar to results in adult ALL (Kantarjian H et al, N Engl J Med 376:836-847, 2017). Also, with blinatumomab, consolidation with subsequent alloSCT showed improved results (12-month RFS rates in patients with vs. without subsequent alloSCT: 70% vs. 30%, respectively) (Locatelli F et al, Blood 136:24-25, 2020). In addition, remission rates with blinatumomab were higher among pediatric patients with low baseline tumor burden (<50% blasts at baseline; 56% CR) versus high tumor burden (≥50% blasts at baseline; 33% CR) (von Stackelberg A et al., J Clin Oncol 34:4381-4389, 2016). In CLINICAL TRIAL-4, no clear association between remission rate and baseline bone marrow blasts was evident, as CR rates were 89%, 25%, 100%, and 50% in patients with baseline tumor burdens of >5% to ≤25%, >25% to ≤50%, >50% to ≤75%, and >75% to ≤100% blasts, respectively. However, interpretation is limited by the small number of patients in each quartile, as well as the relatively high median tumor burden at baseline (Figure 12). This is consistent with another pediatric and young adult study using CD19-targeted CAR T-cell therapy, with no difference in response rates based on disease burden (Gardner RA et al., Blood 129:3322-3331, 2017). Data from CLINICAL TRIAL-4 suggest that KTE-X19 has the potential to provide more favorable efficacy in patients with high disease burden compared to results reported with blinatumomab. In CLINICAL TRIAL-4, a trend toward lower CR+CRi rates appeared to be observed in patients who received prior blinatumomab.

The AE profile in CLINICAL TRIAL-4 was consistent with prior studies of anti-CD19 CAR T cell therapies. For the 24 patients who received KTE-X19, the median time from leukapheresis to delivery to the study site was 16.5 days. In comparison, tisagenlecleucel has a median throughput time of 23 days from receipt of leukapheresis product to delivery to the study site (Tyagarajan S et al., Mol Ther Methods Clin Dev 16:136-144, 2020). The rapid turnaround time of treated patients in CLINICAL TRIAL-4 supports feasibility in the setting of rapidly growing ALL. Once RP2D was established, CLINICAL TRIAL-4 progressed to the Phase 2 portion of the study.

Unmet medical need in R/R pediatric ALL is highest for patients with early relapse or first-line resistant disease with 5-year OS rates of 21%-28% (Sun W et al., Leukemia 32:2316-2325,2018; Crotta A et al., Curr Med Res Opin 34:435-440,2018; Nguyen K et al., Leukemia 22:2142-50,2008; Rheingold SR et al., Journal of Clinical Oncology 37:10008-10008,2019; Oskarsson T et al. al., Haematologica 101:68-76,2016; Schrappe M et al., N Engl J Med 366:1371-81,2012). In addition, the risk of treatment-related morbidity and mortality is 3-5 times higher in patients with MRD-positive disease at the end of first and subsequent lines of therapy than in patients with undetectable MRD. 3 To address this evolving unmet medical need, CLINICAL TRIAL-4 was further modified to broaden the eligibility criteria to include patients with MRD-positive disease as well as those with early first relapse (≤18 months). Additionally, a second cohort was opened to pediatric patients with R/R NHL (diffuse large B-cell lymphoma, Burkitt lymphoma, and primary mediastinal B-cell lymphoma).

Example 5
An open-label, global, multicenter, Phase 3 study was conducted to evaluate the safety and efficacy of axicabtagene siloleucel versus the current standard of care (platinum-based salvage combination chemotherapy regimen followed by high-dose therapy and autologous stem cell transplant in patients responding to salvage chemotherapy) as second-line therapy in adult patients with relapsed or refractory diffuse large B-cell lymphoma (DLBCL). In this study, 359 patients were randomized (1:1) to receive a single infusion of axicabtagene siloleucel or the current second-line standard of care. The primary endpoint was event-free survival (EFS), defined as the time from randomization to the earliest date of disease progression according to the Lugano classification (see Cheson et al, J Clin Oncol. 2014 Sep 20;32(27):3059-68), initiation of new lymphoma treatment, or death from any cause. Key secondary endpoints include objective response rate (ORR) and overall survival (OS). Other secondary endpoints include modified event-free survival, progression-free survival (PFS), and duration of response (DOR). Patients enrolled in the study ranged in age from 22 to 81 years, with 30% over 65 years of age. The study described in this example evaluated a single infusion of axicabtagene siloleucel cell therapy compared to second-line standard of care (SOC) in adult patients with relapsed or refractory LBCL. The study SOC arm was a two-step process: immunochemotherapy was reintroduced after the first relapse, and if the patient responded and could tolerate further treatment, they moved on to high-dose chemotherapy plus stem cell transplant.

Key inclusion criteria:
1. Histologically proven large B-cell lymphoma, including the following types as defined by WHO 2016 (see Swerdlow et al Blood. 2016 May 19;127(20):2375-90. doi:10.1182/blood-2016-01-643569. Epub 2016 Mar 15. Review.)
DLBCL, nonspecific type (ABC/GCB)
HGBL with or without MYC and BCL2 and/or BCL6 rearrangements
DLBCL arising from FL
T-cell/histiocyte-rich large B-cell lymphoma Chronic inflammation-associated DLBCL,
Primary cutaneous DLBCL, lower limb type,
Epstein-Barr virus (EBV)-positive DLBCL
2. Relapsed or Refractory Disease Following First-Line Chemoimmunotherapy Refractory disease is defined as disease that does not show a complete response to first-line therapy (individuals who are intolerant to first-line therapy are excluded).
Progression progression (PD) as best response to first-line treatment
Stable disease (SD) as best response after at least four cycles of first-line therapy (e.g., four cycles of R-CHOP)
Partial response (PR) as best response after at least 6 cycles and biopsy-proven residual disease or disease progression within 12 months of treatment Recurrent disease defined as complete response to first-line treatment followed by biopsy-proven relapse within 12 months of first-line therapy 3. Individuals must have received appropriate first-line therapy including at least:
3. Anti-CD20 monoclonal antibody, and anthracycline-containing chemotherapy unless the tumor is determined by the investigator to be CD20 negative 4. No known or suspected central nervous system involvement by lymphoma Eastern Cooperative Oncology Group (ECOG) performance status of 5.0 or 1 6. Adequate bone marrow function as evidenced by:
Absolute neutrophil count (ANC) of 1000 cells/uL or more
Platelets ≥ 75,000/uL Absolute lymphocyte count ≥ 100/uL 7. Adequate renal, hepatic, cardiac and pulmonary function as evidenced by:
Creatinine clearance of 60 mL/min or more (Cockcroft-Gault formula)
Serum alanine aminotransferase/aspartate aminotransferase (ALT/AST) less than 2.5 times the upper limit of normal (ULN)
Total bilirubin ≤ 1.5 mg/dL Cardiac ejection fraction ≥ 50%, no evidence of pericardial effusion as determined by echocardiogram (ECHO), and no clinically significant electrocardiogram (ECG) findings No clinically significant pleural effusion Baseline oxygen saturation > 92% on room air

The main exclusion criteria were:
1. History of malignancy other than skin cancer other than melanoma or carcinoma in situ (e.g., cervical, bladder, breast) unless disease-free for at least 3 years 2. 2 or more prior lines of treatment for DLBCL 3. Prior autologous or allogeneic stem cell transplant 4. Current history of fungal, bacterial, viral, or other infection that is uncontrolled or requires intravenous antibiotics for management.
5. History of human immunodeficiency virus (HIV) or hepatitis B virus (HBsAg positive) or hepatitis C virus (anti-HCV positive) infection. If previously treated for hepatitis B or C, viral load must be undetectable by quantitative polymerase chain reaction (PCR) and/or nucleic acid testing.
6. Individuals with detectable malignant cells in the cerebrospinal fluid or known brain metastases, or individuals with a history of malignant cells in the cerebrospinal fluid or brain metastases.
7. History or current history of non-malignant central nervous system (CNS) disease, e.g., stroke disorder, cerebrovascular ischemia/hemorrhage, dementia, cerebellar disease, or any autoimmune disease with CNS involvement 8. Presence of any indwelling lines or drains. Dedicated central venous catheters, e.g., Port-a-Cath or Hickman catheters, are acceptable.
9. History of myocardial infarction, cardiac angioplasty or cardiac stenting within 12 months of enrollment, unstable angina, congestive heart failure class II or higher according to the New York Heart Association, or other clinically significant cardiac disease. 10. History of symptomatic deep vein thrombosis or pulmonary embolism within 6 months of enrollment. 11. History of autoimmune disease requiring systemic immunosuppressants and/or systemic disease-modifying agents within the last 2 years. 12. Previous anti-CD19 or CAR-T treatment or previous randomization.

The primary analysis of the study demonstrated superiority of axicabtagene siloleucel compared with standard of care (SOC) in second-line relapsed or refractory large B-cell lymphoma (LBCL). The study achieved its primary endpoint of event-free survival (EFS; hazard ratio 0.398, p<0.0001) and key secondary endpoint of objective response rate (ORR). An interim analysis of overall survival (OS) showed a trend in favor of axicabtagene siloleucel, but the data are immature and further analysis and/or testing may be required.

Safety outcomes from the study were consistent with the known safety profile of axicabtagene siloleucel in the treatment of LBCL in the third-line setting. Six percent of patients experienced grade 3 or higher CRS and 21% experienced grade 3 or higher neurological events. No new safety concerns were identified in this second-line setting.

A validated flow cytometry method was used to assess T phenotype in the Axicabtagene Siloleucel products from this study, analyzing CCR7 and CCR 45 RA, known surface markers of T cell differentiation. Metrics were analyzed by grade of cytokine release syndrome (CRS) and neurological events. P values were obtained by Kruskal-Wallis test.

Table 22 shows the total number of infused central memory T cells (CCR7+CD45RA-) associated with the occurrence of CRS events of grade 2 or higher (compared to grade 1 and no events), while Table 23 shows the total number of infused effector memory and effector T cells (CCR7-) associated with the occurrence of neurological events of grade 3 or higher (compared to grades 2, 1, and no events). These data reveal that different T subsets in axicabtagene siloleucel are differentially associated with toxicity.

Example 6
This example presents further and follow-up results from Example 4 above.

Phase 1 investigated two dose levels and formulations. The primary endpoint was the incidence of dose-limiting toxicities (DLTs). Of 31 enrolled patients, 24 received KTE-X19 (median age 13.5 years, range 3-20 years; median follow-up 36.1 months). No DLTs were observed. All treated patients had grade 3 or higher adverse events, typically hypotension (50%) and anemia (42%). The rates of grade 3 cytokine release syndrome were 33%, ⁷⁵ %, 27%, and 22% across all treatment arms at 2x106, ^1x106 (68 mL formulation), and ^1x106 (40 mL formulation) CAR T cells/kg, with 21%, 25%, 27%, and 11% of patients, respectively, experiencing grade 3 or higher neurological events. Overall complete remission (CR) rates (including CR with incomplete hematologic recovery) were ⁶⁷ %, 75%, 64%, and 67% in all treatment arms with 2x106, ^1x106 (68 mL), and ^1x106 (40 mL) CAR T cells/kg, respectively. Overall minimal residual disease (MRD) negativity was 100% among responders, with 88% of responders undergoing subsequent allogeneic stem cell transplantation (alloSCT). Median duration of remission censored at alloSCT and median overall survival were not reached in the ^1x106 (40 mL) arm (recommended Phase 2 dose). Pediatric/adolescent patients with R/R B-ALL achieved high MRD-negative remission rates with manageable safety following a single dose of KTE-X19. Phase 2 is ongoing at a dose of ^1x106 CAR T cells/kg (40 mL).

Results Patients Thirty-one patients were enrolled and underwent leukapheresis between Feb. 17, 2016 and Aug. 1, 2018. The median time from leukapheresis to KTE-X19 product release was 14.0 days (range, 9.0-20.0) for all treated patients, 16.5 days (range, 12.0-23.0) from leukapheresis to delivery to study site, and 27.0 days (range, 18.0-41.0) from leukapheresis to infusion. Of the 31 enrolled patients, 24 (77%) received conditioning chemotherapy and were subsequently dosed. Seven patients were not dosed for the following reasons: adverse events (AEs; n=1), product failure (n=3), ineligible due to AEs (n=1), product failure and ineligible (n=1), and death (n=1). Twenty-four patients received conditioning chemotherapy followed by KTE-X19. Four patients received the ^2x106 CAR T cells/kg dose, 11 received the ^1x106 CAR T cells/kg (68 mL) dose formulation, and 9 received the ^1x106 CAR T cells/kg (40 mL) dose formulation. Median follow-up of all treated patients was 36.1 months (range, 24.0-53.9). Median age of treated patients was 13.5 years (range: 3-20), 42% of patients had received 3 or more prior lines of therapy, 29% had first-line refractory disease, 25% were R/R after alloSCT, and median bone marrow blasts at screening was 44% (range, 6-99). Prior to enrollment, six (25%) patients had received up-front allo-SCT, eight (33%) had received up-front blinatumomab (including three (13%) who had received blinatumomab as their last up-front therapy), and one (4%) had extramedullary disease. Of the 31 enrolled patients, 30 (97%) received bridging therapy per protocol, with a new baseline disease assessment performed immediately prior to lymphodepleting chemotherapy.

Safety: Among the 3 DLT-evaluable patients who received ^2x106 CAR T cells/kg, no DLTs were observed. All treated patients (n=24) experienced at least one Grade ≥ 3 AE, most commonly hypotension (50%) and anemia (42%). Serious AEs of any grade occurred in 71% of patients. Grade ≥ 3 infections occurred in 42% of patients.

CRS was reported in 21 (88%) of 24 treated patients, with 8 (33%) experiencing grade 3 or higher CRS by modified Lee grading criteria. 33 No grade 4 or grade 5 CRS events occurred. The most common grade 3 or higher CRS symptoms were hypotension (50%) and fever (25%). Any grade and grade 3 or higher hypoxia were observed in 13% and 8% of patients, respectively. The median time to onset of CRS and duration after KTE-X19 infusion were 5 days (range, 1-14) and 7 days, respectively, and all events resolved.

Among all treated patients, NE of any grade was reported in 16 patients (67%), with grade 3 or higher events occurring in 5 patients (21%), with encephalopathy (13%) being the most common grade 3 or higher event. One fully reversible grade 4 NE occurred (cerebral edema) in a patient who received ^1x106 CAR T cells/kg (68 mL). To manage this event, the patient was treated with dexamethasone, mannitol, sodium chloride, and tocilizumab. There were no grade 5 NE. Overall, the median time to onset of NE was 9.5 days (range, 3-60) after infusion, the median time from resolution of first CRS to onset of first NE was 4 days (range, -3-52 [in 4 patients, first CRS resolved after the first NE episode]), and the median duration of NE was 8 days. NE resolved in 14 of 16 patients (88%). The remaining 2 patients' NE was ongoing at the time of death due to an AE (n=1) or progressive disease (n=1). Ten of the 16 patients (63%) who experienced NE had concurrent CRS.

Of all treated patients, 42% received steroids, 63% received tocilizumab, and 46% received vasopressors. Improved overall safety was observed in the nine patients treated with a dose of ^1x106 CAR T cells/kg (40 mL) under the revised toxicity management compared with the four patients treated with ^2x106 CAR T cells/kg and the 11 patients treated with ^1x106 CAR T cells/kg ( ⁶⁸ mL) under the original guidelines. Of patients receiving ^2x106 CAR T cells/kg, 75% experienced grade ≥3 CRS compared with 27% and 22% receiving 1x106 CAR T cells/kg (68 mL and 40 mL, respectively). Grade ≥ 3 NE was observed in 25% of patients receiving ^2x106 CAR T cells/kg and 27% of patients receiving ^1x106 CAR T cells/kg (68 mL), with the lowest incidence (11%) in patients receiving ^1x106 CAR T cells/kg (40 mL). In addition, the median time to onset of NE and CRS appeared to be delayed in the ^1x106 CAR T cells/kg dose cohort compared to the ^2x106 CAR T cells/kg dose cohort.

Of the 8 patients (33%) who died on study, 6 died from progressive disease (median 190.5 days post-KTE-X19 infusion) and 2 patients died from AEs considered unrelated to KTE-X19 (non-grade 5 B-ALL), including disseminated mucormycosis (n=1, 15 days post-KTE-X19 infusion) and E. coli sepsis (n=1, 409 days post-KTE-X19 infusion). Of the patients who died, 3 patients received ^2x106 CAR T cells/kg, 4 received ^1x106 CAR T cells/kg (68 mL), and 1 received ^1x106 CAR T cells/kg (40 mL). No patients tested positive for replicating retrovirus or antibodies to the anti-CD19 CAR at any time point.

Efficacy At a median follow-up of 36.1 months (range, 24.0-53.9), all treated patients (n=24) were evaluable for efficacy. The investigator-assessed overall remission rate was 67%, with 29% of patients (n=7) achieving CR and 38% achieving CRi (n=9). CR+CRi rates were 75%, 64%, and 67% in the ^2x106 , ^1x106 (68 mL), and ^1x106 (40 mL) CAR T cells/kg dose groups, respectively. The median time from infusion to CR+CRi across dose levels was 30 days (range, 26-113 days). The overall MRD negativity rate was 100% among the 16 patients with CR+CRi. Overall, ¹⁶ patients (67%) underwent alloSCT as subsequent consolidation therapy, including 2, 8, and 6 patients in the 2x106, ^1x106 (68 mL), and ^1x106 (40 mL) CAR T cells/kg dose groups, respectively. Fourteen of the 16 patients (88%) who achieved CR+CRi (2, ⁷ , and 5 patients in the 2x106, ^1x106 [68 mL], and ^1x106 [40 mL] CAR T cells/kg dose groups, respectively) underwent subsequent alloSCT. In addition, both patients who achieved a CR with partial hematologic recovery (n=1) and a CR with ablastic hypoplastic/aplastic bone marrow (n=1) proceeded to alloSCT and subsequently achieved a CR. The median time to transplant for all treated patients was 2.3 months (range, 1.4-24.9) after KTE-X19. Of the two patients who achieved CR+CRi but did not undergo subsequent alloSCT, one died of progressive disease and one was lost to follow-up.

The median DOR for the 16 patients who achieved CR+CRi after KTE-X19 was 7.2 months (95% CI, 4.1-14.2) after censoring of subsequent alloSCT, and was not reached at 4.1 and 10.7 months in the ^2x106 , ^1x106 (68 mL), and ^1x106 (40 mL) CAR T cells/kg dose groups, respectively. The median DOR was 14.2 months (95% CI, 3.9-not estimable), with no subsequent censoring of alloSCT and reinitiation of tyrosine kinase inhibitors. The median DOR among the 14 patients with CR+CRi who received subsequent alloSCT after KTE-X19 was 10.7 months (95% CI, 7.2-14.2). Median RFS for all treated patients (n=24) was 5.2 months (95% CI, 0.0-17.8). Median RFS for the 1×10 ⁶ CAR T cells/kg (40 mL) group was not reached, being 5.2 months (95% CI, 0.0-5.2) and 9.1 months (95% CI, 0.0-17.8) for the 2×10 ⁶ and 1×10 ⁶ (68 mL) cells/kg cohorts, respectively. Median RFS among the 16 patients who proceeded to subsequent alloSCT was 9.1 months (95% CI, 9.1-17.8). Median OS was not reached among all treated patients and in both 1×10 ⁶ CAR T cells/kg dose groups, being 8.0 months for the 2×10 ⁶ CAR T cells/kg dose group. The 24-month OS rate was 87.5% (95% CI, 38.7-98.1) for the 1 x ¹⁰⁶ cells/kg (40 mL) dose and 72.7% (95% CI, 37.1-90.3) for the 1 x ¹⁰⁶ cells/kg (68 mL) dose. Overall, as of the data cut-off date, 8 (33%) of the 24 treated patients had died, 1 had discontinued due to withdrawal of consent, and 1 was lost to follow-up. The remaining 14 (58%) patients were still alive and under continued follow-up as of the data cut-off date, all of whom underwent subsequent alloSCT after KTE-X19.

Based on safety and efficacy data analysis, the RP2D was 1 x ¹⁰⁶ KTE-X19 cells/kg (40 mL formulation) with revised toxicity controls.

Translational Analysis CAR T cell proliferation in peripheral blood, measured by polymerase chain reaction (PCR) and expressed as CAR gene copies/μg DNA in blood, was observed across dose groups, with peak CAR T cell levels reached by day 14, followed by CAR T cell shrinkage to baseline thereafter. Median CAR T cell levels were undetectable in blood by PCR across all dose groups 3 months after KTE-X19 infusion. Median peak CAR gene copies/μg DNA in blood was similar between 1×10 ⁶ CAR T cells/kg dose cohorts, but higher in the 2×10 ⁶ CAR T cells/kg cohort. Patients who achieved CR+CRi trended toward higher median peak blood CAR gene copies/μg DNA in blood than non-responders, as did MRD-negative patients compared to MRD-positive patients. The CAR gene copy number/μg DNA in blood tended to be higher in patients with grade 3 or higher NE compared with patients with grade 2 or lower NE. However, with this limited sample size, there was no clear difference in the peak CAR gene copy number/μg DNA in blood in patients with either high-grade or low-grade CRS. The median peak CAR gene copy number/μg DNA in blood was 5.16×10 ⁴ (range, 0-2.40×10 ⁵ ) in 16 patients who did not receive upfront blinatumomab and 6.15×10 ³ (range, 0-2×49×10 ⁵ ) in 8 patients who received upfront blinatumomab.

Peak levels of multiple key serum cytokines, chemokines, and pro-inflammatory biomarkers occurred by day 7. Proportional to peak CAR proliferation, several serum analytes tended to be higher in patients receiving 2×10 ⁶ CAR T cells/kg compared to 1×10 ⁶ CAR T cells/kg (interleukin [IL]-2, IL-5, IL-6, IL-8, IL-10, IL-15, IL-16, ferritin, granzyme B, intercellular adhesion molecule 1 [ICAM-1], interferon gamma [IFN-γ], and tumor necrosis factor alpha [TNF-α]).

Peak serum levels of the analytes VCAM-1 and IL-16 were associated with grade ≥3 CRS. No such association was observed in subjects with ≥3 NE. This may have been due to the small number of patients with ≥3 NE. Product characteristics were similar across dose levels. Levels of less differentiated CCR7+ T cells in the product were higher in CR+CRi patients and tended to be higher in MRD-negative patients. This product profile also appeared to be associated with higher levels of neurotoxicity, but was not associated with CRS. The ratio of CD4 to CD8 T cells was not associated with response or toxicity.

Discussion In Phase 1 of Clinical Trial-4, no DLTs were observed with KTE-X19 among DLT-evaluable pediatric or adolescent patients with R/R B-AL. No DLTs were observed with the initial dose of ^2x106 CAR T cells/kg, but to further improve the risk:benefit ratio, a lower dose of ^1x106 CAR T cells/kg with a 68 mL formulation was explored in a second cohort of patients, and dosing and toxicity management was further optimized with a 40 mL formulation and revised toxicity management in a third cohort of ^1x106 CAR T cells/kg. This resulted in a more optimal risk:benefit ratio for the ^1x106 CAR T cells/kg (40 mL) dose level, with notable improvements in CRS and NE. In addition, the MRD-negativity rate was 73% or higher for all formulations, but the MRD-negativity rate and CR-only rate were highest in patients receiving ^1x106 CAR T cells/kg (40 mL). Importantly, median DOR, RFS, and OS were not reached among the nine patients in the ^1x106 CAR T cells/kg (40 mL) cohort, with most responders (5/6 [83%]) proceeding to subsequent allo-SCT. Recognizing the limitations of the small cohort, the 24-month OS rate in this group was nevertheless 87.5%. These results potentially suggest significant durability of response with optimized dosing/formulation of KTE-X19 followed by allo-SCT in pediatric/adolescent patients with R/R B-ALL.

The role of alloSCT after anti-CD19 CAR T-cell therapy in pediatric/adolescent patients with R/R B-ALL is not yet fully defined, and studies in adult populations have provided somewhat conflicting results. In this study, the median DOR censored at subsequent alloSCT and OS was not reached in patients treated with RP2D at ^1x106 CAR T-cells/kg (40 mL). Fourteen of 16 patients (88%) who achieved CR+CRi, including five treated with RP2D, received alloSCT as subsequent therapy. AlloSCT was not required according to the protocol but was allowed at the investigator's discretion. Clinical Trial-4 was not designed to evaluate outcomes after subsequent therapy. However, most responding patients proceeded to alloSCT after KTE-X 19 as per the investigator's decision.

Evaluation of DOR in Clinical Trial-4 without censoring of subsequent treatments, including alloSCT, revealed a favorable median of 14.2 months. In addition, the median RFS with censoring of subsequent alloSCT was 5.2 months, whereas the uncensored value was 9.1 months. It has previously been reported that children and young adults with R/R CD19+ ALL who were not alloSCT-naive but who received consolidation alloSCT after anti-CD19 CAR T-cell therapy showed a trend toward improved leukemia-free survival with ≥1 year of follow-up. In a recently published phase 1 study of anti-CD19 CAR T-cell therapy in children and young adults with R/R B-ALL, in which 75% of MRD-negative responders proceeded to alloSCT, the median OS was 70.2 months after alloSCT at 4.8 years of follow-up, the 5-year event-free survival rate after alloSCT was 61.9%, and the cumulative recurrence rate after alloSCT was only 9.5%. Interestingly, a retrospective review in children and young adults found that CD34-selected T-cell-depleted alloSCT after CAR T-cell therapy could result in improved transplant-related mortality and OS compared to those with unmodified alloSCT.

The data presented herein support a promising potential role for KTE-X19 in prolonging durability of response and survival in children/adolescents with R/R B-ALL, especially when followed by alloSCT.

Although differences in study design and patient populations preclude direct interstudy comparisons, a recent study with blinatumomab, which also targets CD19, shows a median OS of only 7.7 months in pediatric R/R B-ALL, similar to results in adult ALL. 40 Also, for blinatumomab, consolidation with subsequent alloSCT showed improved outcomes (12-month RFS rates of 70% vs. 30% for patients with vs. without subsequent alloSCT, respectively). In addition, remission rates with blinatumomab were higher among pediatric patients with low baseline tumor burden (<50% blasts; 56% CR) versus high baseline tumor burden (≥50% blasts; 33% CR). In CLINICAL-4, no clear association between remission rate and baseline bone marrow blasts was evident. This is because CR rates were 89%, 25%, 100%, and 50% in patients with >5% to ≤25%, >25% to ≤50%, >50% to ≤75%, and >75% to ≤100% blasts at baseline, respectively. This is consistent with another pediatric and young adult study using CD19-targeted CAR T-cell therapy, with no difference in response rates based on disease burden. Data from CLINICAL-4 suggest that KTE-X19 has the potential to provide more favorable efficacy in patients with high disease burden compared to results reported with blinatumomab. In CLINICAL-4, a trend toward lower CR+CRi rates appeared to be observed in patients who received prior blinatumomab.

The AE profile in CLINICAL-4 was consistent with prior studies of anti-CD19 CAR T-cell therapies. For patients receiving KTE-X19, the median time from leukapheresis to delivery to the study site was 16.5 days. In comparison, for the first 37 commercially manufactured tisagenlecleucel products for B-ALL patients, the reported median throughput time was 23 days from receipt of leukapheresis product to delivery to the clinical site. The rapid turnaround time for treated patients in CLINICAL-4 supports feasibility in the setting of rapidly growing ALL.

Unmet medical need in R/R pediatric ALL is highest in patients who relapse early or who have first-line refractory disease with 5-year OS rates of 21% to 28%. 2,4,8,43-45 Additionally, the risk of treatment-related morbidity and mortality is 3-5 times higher in patients with MRD-positive disease at the end of first and subsequent lines of therapy than in patients with undetectable MRD3.

To address this evolving unmet medical need, Clinical Trial-4 was further modified to broaden eligibility criteria to include patients with MRD-positive disease and those with early first relapse (within 18 months). Additionally, a second cohort was opened to pediatric patients with R/R NHL (diffuse large B-cell lymphoma, Burkitt lymphoma, and primary mediastinal B-cell lymphoma). Of the investigational agents in US Food and Drug Administration/European Medicines Agency-approved or registrational trials in pediatric R/R B-ALL, Clinical Trial-4 is the first CAR T-cell therapy trial to report >3 years of follow-up with positive results.

All publications, patents, patent applications, and other documents cited in this application are incorporated herein by reference in their entirety for all purposes to the same extent as if each individual publication, patent, patent application, or other document was individually indicated to be incorporated by reference for all purposes. While various specific embodiments/aspects have been illustrated and described, it will be understood that various changes can be made without departing from the spirit and scope of the disclosure.

Secondary Malignancies In some embodiments, patients treated with immune cells (e.g., CAR T cells) or other genetically modified autologous T cell immunotherapies (e.g., targeting CD19) may develop secondary malignancies. In certain embodiments, patients treated with CAR T cells (e.g., targeting CD19) or other genetically modified allogeneic T cell immunotherapies may develop secondary malignancies. In some embodiments, the method includes lifelong monitoring for secondary malignancies.
[Item 1]
1. A method for treating relapsed/refractory B-cell precursor acute lymphoblastic leukemia in a subject, comprising administering to the subject a therapeutically effective amount of immune cells against a tumor antigen, wherein the subject is a pediatric or adolescent subject.
[Item 2]
Item 2. The method according to item 1, wherein the therapeutically effective amount of immune cells is about 1 x 10 ⁶ to about 2 x 10 ⁶ immune cells per kg of body weight.
[Item 3]
3. The method of claim 2, wherein the immune cells are administered in a total volume of about 40 mL to 68 mL.
[Item 4]
3. The method of claim 2, wherein the immune cells are administered in a total volume of about 40 mL.
[Item 5]
Item 2. The method of item 1 , wherein the therapeutically effective amount of immune cells is about 1 x 10 ⁶ immune cells per kg of body weight.
[Item 6]
2. The method of claim 1, wherein the immune cells are administered as a first, second, third, fourth, fifth, or sixth line treatment or prior to disease progression.
[Item 7]
The tumor antigens include tumor associated surface antigens, 5T4, alpha fetoprotein (AFP), B7-1 (CD80), B7-2 (CD86), BCMA, B-human chorionic gonadotropin, CA-125, carcinoembryonic antigen (CEA), CD123, CD133, CD138, CD19, CD20, CD22, CD23, CD24, CD25, CD30, CD33, CD34, CD4, CD40, CD44, CD56, CD8, CLL-1, c- Met, CMV-specific antigen, CS-1, CSPG4, CTLA-4, DLL3, disialoganglioside GD2, ductal epithelial mucin, EBV-specific antigen, EGFR variant III (EGFRvIII), ELF2M, endoglin, ephrinB2, epidermal growth factor receptor (EGFR), epithelial cell adhesion molecule (EpCAM), epithelial tumor antigen, ErbB2 (HER2/neu), fibroblast-associated protein (fap), FLT3, folate-binding protein , GD2, GD3, glioma-associated antigens, glycosphingolipids, gp36, HBV-specific antigens, HCV-specific antigens, HER1-HER2, HER2-HER3 combinations, HERV-K, high molecular weight melanoma-associated antigen (HMW-MAA), HIV-1 envelope glycoprotein gp41, HPV-specific antigens, human telomerase reverse transcriptase, IGF I receptor, IGF-II, IL-11Rα, IL-13R-a2, influenza virus-specific specific antigens; CD38, insulin growth factor-1 (IGF1), intestinal carboxylesterase, kappa chain, LAGA-1a, lambda chain, Lassa virus specific antigen, lectin-reactive AFP, lineage-specific antigens or tissue-specific antigens, e.g., CD3, MAGE, MAGE-A1, major histocompatibility complex (MHC) molecules, major histocompatibility complex (MHC) molecules presenting tumor-specific peptide epitopes, M-CSF, melanoma-associated antigen, mesothelin, MN-CA IX, MUC-1, mutant hsp70-2, mutant p53, mutant ras, neutrophil elastase, NKG2D, Nkp30, NY-ESO-1, p53, PAP, prostase, prostate specific antigen (PSA), prostate cancer tumor antigen-1 (PCTA-1), prostate specific antigen protein, STEAP1, STEAP2, PSMA, RAGE-1, ROR1, RU1, RU2 (AS), surface adhesion molecules, survivin and telomerase, TAG-72, extra domain A (EDA) and extra domain B (EDB) of fibronectin and A1 domain of tenascin-C (TnC A1), thyroglobulin, tumor stromal antigen, vascular endothelial growth factor receptor-2 (VEGFR2), virus specific surface antigens, such as HIV specific antigens (e.g., HIV gp120), as well as any derivative or variant of these surface antigens.
[Item 8]
8. The method of claim 7, wherein the target antigen is CD19.
[Item 9]
The method further comprises preconditioning the subject with one or more preconditioning agents, wherein the one or more preconditioning agents are selected from at least one of an alkylating agent and a platinum-based agent, the alkylating agent being selected from melphalan, chlorambucil, cyclophosphamide, mechlorethamine, mustine (HN2), uramustine, uracil mustard, melphalan, chlorambucil, ifosfamide, bendamustine, carmustine, lomustine, streptozocin, Item 9. The method of item 8, wherein the platinum-based preconditioning agent is selected from the group consisting of platinum, cisplatin, carboplatin, nedaplatin, oxaliplatin, satraplatin, triplatin tetranitrate, procarbazine, altretamine, triazene, dacarbazine, mitozolomide, temozolomide, dacarbazine, temozolomide, and any combination thereof.
[Item 10]
10. The method of claim 9, wherein the preconditioning agent comprises cyclophosphamide and fludarabine.
[Item 11]
10. The method of claim 9, wherein the cyclophosphamide is administered at a dose of 200 mg/m2/day to 2000 mg/m2/day and the fludarabine is administered at a dose of 20 mg/m2/day to 900 mg/m2/day.
[Item 12]
10. The method of claim 9, wherein administration of the one or more preconditioning agents is initiated at least 7 days, at least 6 days, at least 5 days, at least 4 days, at least 3 days, at least 2 days, or at least 1 day prior to administration of the immune cells.
[Item 13]
The method of claim 1, wherein the subject has a high tumor burden.
[Item 14]
2. The method of claim 1, further comprising at least one of administering tocilizumab for management of neurological events only in the setting of cytokine release syndrome, and administering a corticosteroid for management of grade 2 neurological events.
[Item 15]
The method of claim 1, wherein the subject is at high risk of disease progression, and the subject is at high risk if the subject exhibits disease progression within 24 months of initial diagnosis.
[Item 16]
2. The method of claim 1, wherein the immune cells are selected from tumor infiltrating lymphocytes (TIL), NK cells, autologous T cells, allogeneic T cells, and genetically engineered autologous T cells (eACT), and any combination thereof.
[Item 17]
2. The method of claim 1, wherein the immune cell is a CAR T cell.
[Item 18]
1. A method of treating cancer in a subject in need thereof, wherein the cancer is non-Hodgkin's lymphoma (NHL) or relapsed/refractory B-cell precursor acute lymphoblastic leukemia or relapsed/refractory B-cell non-Hodgkin's lymphoma (R/R B-ALL), comprising administering to the subject a therapeutically effective amount of immune cells directed against a tumor antigen, wherein the immune cells are autologous T cells expressing an anti-CD19 chimeric antigen receptor (CAR).
[Item 19]
20. The method of claim 18, wherein the cancer is NHL, and the NHL is mantle cell lymphoma (MCL) or indolent NHL (iNHL).
[Item 20]
20. The method of claim 19, wherein the iNHL is marginal zone lymphoma (MZL) or follicular lymphoma (FL).
[Item 21]
20. The method of claim 19, wherein the cancer is NHL, and the NHL is (relapsed or refractory) diffuse large B-cell lymphoma (DLBCL) - nonspecific type, primary mediastinal large B-cell lymphoma, high-grade B-cell lymphoma, or DLBCL arising from follicular lymphoma.

Claims (21)

A method for treating relapsed/refractory B-cell precursor acute lymphoblastic leukemia in a subject, comprising administering to the subject a therapeutically effective amount of immune cells against a tumor antigen, the subject being a pediatric or adolescent subject. 2. The method of claim 1, wherein the therapeutically effective amount of immune cells is about 1 x ¹⁰ to about 2 x ¹⁰ immune cells per kg of body weight. The method of claim 2, wherein the immune cells are administered in a total volume of about 40 mL to 68 mL. The method of claim 2, wherein the immune cells are administered in a total volume of about 40 mL. 2. The method of claim 1, wherein the therapeutically effective amount of immune cells is about 1 x ¹⁰⁶ immune cells per kg of body weight. The method of claim 1, wherein the immune cells are administered as a first, second, third, fourth, fifth, or sixth line treatment, or prior to disease progression. The tumor antigens include tumor-associated surface antigens, 5T4, alpha-fetoprotein (AFP), B7-1 (CD80), B7-2 (CD86), BCMA, B-human chorionic gonadotropin, CA-125, carcinoembryonic antigen (CEA), CD123, CD133, CD138, CD19, CD20, CD22, CD23, CD24, CD25, CD30, CD33, CD34, CD4, CD40, CD44, CD56, CD8, CLL-1, c- Met, CMV-specific antigen, CS-1, CSPG4, CTLA-4, DLL3, disialoganglioside GD2, ductal epithelial mucin, EBV-specific antigen, EGFR variant III (EGFRvIII), ELF2M, endoglin, ephrinB2, epidermal growth factor receptor (EGFR), epithelial cell adhesion molecule (EpCAM), epithelial tumor antigen, ErbB2 (HER2/neu), fibroblast-associated protein (fap), FLT3, folate-binding protein , GD2, GD3, glioma-associated antigens, glycosphingolipids, gp36, HBV-specific antigens, HCV-specific antigens, HER1-HER2, HER2-HER3 combinations, HERV-K, high molecular weight melanoma-associated antigen (HMW-MAA), HIV-1 envelope glycoprotein gp41, HPV-specific antigens, human telomerase reverse transcriptase, IGF I receptor, IGF-II, IL-11Rα, IL-13R-a2, influenza virus-specific specific antigens; CD38, insulin growth factor-1 (IGF1), intestinal carboxylesterase, kappa chain, LAGA-1a, lambda chain, Lassa virus specific antigen, lectin-reactive AFP, lineage-specific antigens or tissue-specific antigens, e.g., CD3, MAGE, MAGE-A1, major histocompatibility complex (MHC) molecules, major histocompatibility complex (MHC) molecules presenting tumor-specific peptide epitopes, M-CSF, melanoma-associated antigen, mesothelin, MN-CA IX, MUC-1, mutant hsp70-2, mutant p53, mutant ras, neutrophil elastase, NKG2D, Nkp30, NY-ESO-1, p53, PAP, prostase, prostate specific antigen (PSA), prostate cancer tumor antigen-1 (PCTA-1), prostate specific antigen protein, STEAP1, STEAP2, PSMA, RAGE-1, ROR1, RU1, RU2 (AS), surface adhesion molecules, survivin and telomerase, TAG-72, extra domain A (EDA) and extra domain B (EDB) of fibronectin and A1 domain of tenascin-C (TnC A1), thyroglobulin, tumor stromal antigen, vascular endothelial growth factor receptor-2 (VEGFR2), virus specific surface antigens, such as HIV specific antigens (e.g., HIV gp120), and any derivative or variant of these surface antigens. The method of claim 7, wherein the target antigen is CD19. The method further comprises preconditioning the subject with one or more preconditioning agents, the one or more preconditioning agents being selected from at least one of an alkylating agent and a platinum-based agent, the alkylating agent being melphalan, chlorambucil, cyclophosphamide, mechlorethamine, mustine (HN2), uramustine, uracil mustard, melphalan, chlorambucil, ifosfamide, bendamustine, carmustine, lomustine, streptozocin, sucrose, 9. The method of claim 8, wherein the platinum-based preconditioning agent is selected from the group consisting of platinum, cisplatin, carboplatin, nedaplatin, oxaliplatin, satraplatin, triplatin tetranitrate, procarbazine, altretamine, triazene, dacarbazine, mitozolomide, temozolomide, dacarbazine, temozolomide, and any combination thereof. The method of claim 9, wherein the preconditioning agent comprises cyclophosphamide and fludarabine. 10. The method of claim 9, wherein said cyclophosphamide is administered at a dose of 200 mg/m ² /day to 2000 mg/m ² /day and said fludarabine is administered at a dose of 20 mg/m ² /day to 900 mg/m ² /day. 10. The method of claim 9, wherein administration of the one or more preconditioning agents begins at least 7 days, at least 6 days, at least 5 days, at least 4 days, at least 3 days, at least 2 days, or at least 1 day prior to administration of the immune cells. The method of claim 1, wherein the subject has a high tumor burden. The method of claim 1, further comprising at least one of administering tocilizumab for management of neurological events only in the setting of cytokine release syndrome and administering a corticosteroid for management of grade 2 neurological events. The method of claim 1, wherein the subject is at high risk for disease progression, and the subject is at high risk if the subject exhibits disease progression within 24 months of initial diagnosis. The method of claim 1, wherein the immune cells are selected from tumor infiltrating lymphocytes (TIL), NK cells, autologous T cells, allogeneic T cells, and genetically engineered autologous T cells (eACT), and any combination thereof. The method of claim 1, wherein the immune cells are CAR T cells. A method of treating cancer in a subject in need of such treatment, wherein the cancer is non-Hodgkin's lymphoma (NHL) or relapsed/refractory B-cell precursor acute lymphoblastic leukemia or relapsed/refractory B-cell non-Hodgkin's lymphoma (R/R B-ALL), comprising administering to the subject a therapeutically effective amount of immune cells against a tumor antigen, the immune cells being autologous T cells expressing an anti-CD19 chimeric antigen receptor (CAR). The method of claim 18, wherein the cancer is NHL, and the NHL is mantle cell lymphoma (MCL) or indolent NHL (iNHL). The method of claim 19, wherein the iNHL is marginal zone lymphoma (MZL) or follicular lymphoma (FL). 20. The method of claim 19, wherein the cancer is NHL, and the NHL is (relapsed or refractory) diffuse large B-cell lymphoma (DLBCL) - unspecified type, primary mediastinal large B-cell lymphoma, high-grade B-cell lymphoma, or DLBCL arising from follicular lymphoma.

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